DE69928809T2 - DIFFERENTIAL DIAGNOSIS OF NEURODEGENERATION - Google Patents

Abstract

The present invention relates to new methods for the specific detection, quantification and/or differential diagnosis of neurodegeneration in an individual making use of a combination assay detecting at least three neurological markers in one or more body fluids of said individual, the type and degree of neurodegeneration being reflected in the quantitative changes in the level of all of said neurological markers compared to the control sample. The present invention also relates to methods for the detection of Rab3a, SNAP25 and alpha -synuclein in cerebrospinal fluid and to the use of these methods in a combination assay for specific detection, quantification and/or differential diagnosis of neurodegeneration.

Description

AREA OF INVENTION

The The present invention relates to the field of diagnosis of neurodegeneration. The present invention relates to novel methods for differential diagnosis, using a combination test that is different neurological Markers in body fluids detected. The present description also relates to new methods for detecting Rab3a, SAP25 or an α-synuclein in cerebrospinal fluid and the use of these methods in a combination test for differential diagnosis of neurodegeneration.

BACKGROUND OF THE INVENTION

neurodegeneration is a feature of several neurological disorders. Neurodegeneration can cause axonal damage, gradually developing neuronal death; Anomalies in neurotransmitter release or receptor function; destruction of myelin, changes the bloodstream in the CNS; Dysfunction of the blood-brain barrier and / or changed Oxygen metabolism; Difficulty with other CNS metabolic pathways and / or various other often unknown aspects that are a malfunction of the CNS can cause include. Nowadays different diseases come with different ones Aspects of neuronal dysfunction (for a review see Wilson et al., 1991). For example, Alzheimer's disease is the most important all neurodegenerative diseases involving death and disappearance of nerve cells in the cerebral cortex are involved. this is the common Dementia in the elderly Persons responsible for Patients and families suffer and get an economic loss in the form of costs means for a long - term care of patients are necessary due to the Disease completely are disabled. Dementia of the frontal lobe / temporalis is the second most Type of primary degenerative Dementia and this accounts for about 3 to 10% of all patients with dementia from (Brun, 1993, Knopman, 1993). The clinical picture is through the Presence of a predominant syndrome of the frontal lobe (Sjögren, 1997), that also with other disturbances, like mood disorders and schizophrenia can be observed (Abbruzzese et al., 1997). The Lewy body disease is a condition associated with progressive dementia or psychosis. Parkinson's signs, which are initially absent or weak, eventually become common and the stiffness becomes ordinary heavy. Lewy body be in great Dimensions in the Brainstem, basal forebrain, in hyopthalamic nuclei and neocortex found.

Parkinson's disease is a type of Lewy body disease that is in the middle or late Age with a very gradual Progress and a prolonged Course occurs. It can be considered as an example of a nervous system disorder look at that mainly includes the dopaminergic nigrostriatum system. A cerebrovascular disease is triggered by one of several pathological processes, the the blood vessels of the brain include. It's the third most common Cause for Death after heart disease and cancer in industrialized countries and has a global prevalence of 794 per 100,000. Five Percent of the population is over 65 stroke, an acute neurological injury, which occurs as a result of one of these pathological processes. The Neurodegeneration may also be due to exposure to certain chemical Compounds (Table 1), radiation, chemotherapy or hypoxic-ischemic events be induced. Long-term complications of treatment (or Prophylaxis) of leukemia in childhood and brain tumor include behavioral changes, poor academic performance, memory loss, intellectual Descent, growth retardation, hormonal imbalances and abnormal CT scans (cerebral atrophy, ventricular Dilatation, intracerebral calcifications). Delays of the intellectual Development (deficits in IQ, memory, attention, visuospatial Ability) (Fletcher et al., 1988) or decreases in cognitive function Leukemia survivors (Ochs et al., 1991) were given after radiation or chemotherapy without cranial irradiation observed. In addition, children younger than 4 years are, especially opposite neuroloxic effects of cranial radiotheraphy and / or chemotherapy susceptible (Moore et al., 1986; Jannoun et al., 1983). For the most Agents produce high-dose therapy, combination chemotherapy, accompanying cranial radiotherapy and intracarotic or intrathecal Injections rather neurological complications than oral or intravenous Standard therapy. Any part of the nervous system can be damaged. As cancer patients are treated more aggressively, more chemo get and longer and new chemotherapy drugs are being developed and common means be used more intensively or in new ways become neurological Complications of cancer chemotherapy more common, more severe and more complex.

Most neurological conditions for which the patient seeks general medical care are based on easily grasped disease processes. The task of the physician is the development of a neurological analysis procedure that allows for the exact diagnosis of the site of the disorder and its possible cause leads. Only after a precise diagnosis is it possible to effectively manage and treat the disease. Some techniques for diagnosing neurodegeneration in patients have been developed as Positional Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT) and Nuclear Magnetic Resonance Spectroscopy (NMRS), which allow the study of brain function and structure. However, most neurological diseases are still clinically diagnosed based on the exclusion of other forms of disorders, and in some cases it is even not possible to distinguish between different neurological disorders. The Lewy body type of dementia or Lewy body dementia (LBD), which is sensitive to, for example, neuroleptics, is clinically very difficult to distinguish from Alzheimer's disease (McKeith et al., 1996, Ballard et al., 1998). Most patients (more than 75%) are neuropathologically defined as patients with Alzheimer's disease, whereas an estimated 15 to 25% of clinically diagnosed Alzheimer's disease patients have Lewy body dementia (Hooten et al., 1998). Since Lewy body dementia is more susceptible to acetylcholinesterase treatment, differentiation of Lewy body dementia from Alzheimer's disease is essential for optimizing treatment (Levy et al., 1994, Perry et al., 1994; Wilcock et al., 1994).

moreover Dementia of the frontal lobe / temporalis is often considered other types dementia or other psychiatric disorders misdiagnosed, as the symptoms of dementia of the frontal lobe / temporalis also at other disorders can be observed.

It There is no clear cut difference between vascular disease and Alzheimer's disease, and again, there is a risk of misdiagnosis obviously. As a pharmacological treatment of vascular disease possible is is an early one correct diagnosis crucial.

Also the detection and treatment of brain damage induced by Remedies, such as certain chemical compounds, radiation, chemotherapy or hypoxic-ischemic Events remains a frequent and important clinical Problem for most neurologists. In cases, where the clinical diagnosis is doubtful, a definite Diagnosis irrevocable only by neuropathological examination respectively. As such, a precise differential diagnosis of neurodegeneration only possible post mortem. Therefore, a procedure to early Recording neurological disorders in patients and for monitoring neurological changes, which are induced by various means, very helpful in determining whether an action of the inducing agent is continued Can, whether appropriate doses and drugs in individual patients be used and to start the right treatments.

A series of neurological markers has recently become available, the the conditions of the central nervous system (CNS) in terms of cell death, axon growth and re-induction, inflammation and / or dysfunction of the blood-brain barrier reflect.

The microtubule-associated protein tau, for example, is a major protein component of paired helical filaments (PHF) and neurofibrillary tangles (NFT) (Brion et al., 1985, Delacourt and Defossez, 1986, Grundke-Igbal et al., 1986, Kosik et al. 1986, Wood et al., 1986; Kondo et al., 1988). The protein tau exists in different isoforms, of which 4 to 6 are found in the brain of adults, but only 1 isoform is detected in the fetal brain. Diversity of isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989, Goedert et al., 1989, Andreadis et al., 1992). The most conspicuous feature of the tau protein, as derived by molecular cloning, is a span of 31 or 32 amino acids found in the carboxy-terminal part of the molecule, which can be repeated either three or four times. Additional diversity is created by 29 or 58 amino acid insertions into the NH ₂ -terminal portion of the tau molecules (Goedert et al., 1989). In vivo tau promotes microtubule assembly and stability in the axon compartment of the neurons through interactions, including its microtubule-binding domain, located in the repeated region of tau (255-381) (Lewis et al., 1988). Under normal circumstances, the brain of an adult contains 2 to 3 moles of phosphate per molecule of tau (Selden and Pollard, 1983, Ksiezak-Reding et al., 1992). Phosphorylation of various sites in normal tau, as studied in rats and humans, depends on the developmental stage (Lee et al., 1991, Bramblett et al., 1993, Goedert et al., 1993). Tau variants of 60, 64 and 68 kDa resulting from phosphorylation have been detected in areas of the brain exhibiting neurofibrillary tangles (Delacourte et al., 1990; Goedert et al., 1992; Flament et al., 1990, Greenberg and Davies, 1990). These brains contain 6 to 8 moles of phosphate per mole of tau (Ksiezak-Reding et al., 1992). In Tau isolated from PHF (PHF-Tau), phosphorylation occurs at multiple positions (Igbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992). Heretofore, the detection of phospho-tau in brain extracts, either via antibodies (Mab Alz50, Ghanbari et al., 1990; Mab Ab423: Harrington et al., 1991; Mab AT120: Vandermeeren et al., 1993; Mab AT180; Mab AT270 : Inter National Application, published under WO 95/17429 and Mab AT8: International Application, published under WO 93/08302) or on the change of molecular weight (Flament et al., 1990) or else as a result of functional tests (Bramblett et al., 1992) used to distinguish dementia with altered cytoskeletal properties of normal aged individuals or from patients with other types of dementia. A combination of monoclonal antibodies, each recognizing non-phosphorylated epitopes of tau, was used to detect the presence of tau and PHF tau in cerebrospinal fluid (Van de Voorde et al., 1995).

The The gamma subunit of neuron-specific enolase (NSE) is a Main component of the neuronal cytosol (Kato et al., 1981). NSE illustrates 3% of total soluble brain protein. In adults It is believed that they are useful in evaluating the active neuronal Damage of the ischemic, infectious or tumor origin (Garcia et al., 1994). NSE in serum of children has not been studied. Nara et al., (1988) showed that a high NSE level in cerebrospinal fluid or serum with a poor outcome and death in comatose children. An increased However, serum NSE is not necessarily CNS origin. Some Tissues, including peripheral neurons, endocrine glands, lymphocytes, red blood cells and platelets contain NSE (Kaiser, 1989), which prohibits the use of this marker alone endanger can.

β-amyloid, a 40 to 43 amino acid long peptide, is derived via proteolytic cleavage from a large precursor protein called amyloid precursor protein or APP. Amyloid is produced during the metabolism of normal cells. The amyloid peptide has a high degree of heterogeneity. Two major forms of β-amyloid have been identified, β-amyloid _(1-40) and β-amyloid _(1-42) β-amyloid _(1-42) being a major component of the neuritic plaques of Alzheimer's disease, Down syndrome and normal aged brains. It can be neurotoxic, and is known to increase the vulnerability of neurons to other insults. In addition, low levels of soluble amyloid can induce cholinergic hypoactivity that does not depend on the concomitant neurotoxicity. Acetylcholine plays a crucial role in cognitive processes (Auld et al., 1998). The development of high affinity monoclonal antibodies that specifically recognize well-defined epitopes of the peptide results in a facile test for the β-amyloid _(1-42) peptide in unconcentrated cerebrospinal fluid (Citron et al., 1997, Johnson-Wood et al , 1997). This test may also prove valuable in monitoring long-term effects of drugs, radiation, or chemicals that interfere with APP processing.

Growth-associated protein-43 (GAP-43), also referred to as neuromodulin or B-50, is a nerve tissue-specific protein found primarily in the axons and presynaptic endings. GAP-43 is believed to play a major role in nerve growth, neurite outgrowth, and regeneration and neuronal growth (Skene and Willard, 1981; Basi, 1987; Benowitz et al., 1989; Mercken et al., 1992a) Synapse proteins have different roles the synapse function. Proteins, such as synapsin, are important in determining the amount of vesicles available for fusion, whereas Rab3a and rabphilin are important for delivering the vesicles to the membrane. The docking process is determined by a molecular complex of synaptobrevin, SNAP25, Sec, and syntaxin, where it is believed that CSP and synaptotagmin play an important role in the Ca ²⁺ -dependent release of vesicle content. Alpha-synuclein is abundant in synapses of the substantia nigra and basal ganglia, and belongs to a family of proteins including α-synuclein and γ-synuclein.

Such intracellular Markers in body fluids present and stable and that the metabolic state of the neurons in the central nervous system can be used for the early detection of neurodegeneration, even be suitable for the onset of clinical signs. One biochemical index of neuronal function that may be Can be used in combination with other diagnostic methods very helpful for improving the accuracy of the clinical diagnosis and therapeutic monitoring of neurodegeneration.

Alzheimer's disease is characterized by abundant extracellular senile plaques, intracellular nodules, and loss of synapse. Tau and β-amyloid _(1-42) are essential components of each of these globules and plaques, the two diagnostic structures in the neuropathological study of AD. Both tau and β-amyloid _(1-42) were detected in cerebrospinal fluid (CSF), and it is now well recognized that CSF tau and CSF-β amyloid _{(1-42) are} neurological markers of Alzheimer's disease. Disease, although it is not yet known how changes in CSF levels affect the pathophysiology of Alzheimer's disease. CSF-tau is elevated in Alzheimer's patients compared to age-matched controls and affects the number of balls in the brain, whereas β-amyloid _(1-42) is reduced in Alzheimer's disease. β-amyloid _(1-42) may not be involved in plaque formation since it is reduced in dementia without senile diffuse plaques, such as dementia of the frontal lobe. Although studies on brain tissue suggest a relationship for plaques and certain tangles to the extent of dementia, the levels of CSF-tau and CSF-β-amyloid _(1-42) are inconsistent with the extent of dementia as defined by the mini-mental state, and an overlap with other types of dementia also occurs. The use of β-amyloid _(1-40) as a neurological marker in addition to tau and β-amyloid _(1-42) (Shoji et al., 1998) does not improve the diagnostic test for Alzheimer's disease, as the level of β- Amyloid _(1-40) in Alzheimer's disease patients did not change as compared with the normal controls (Motter et al., 1995).

investigations rat GAP-43 resulted in decreased levels in the endocortex Alzheimer's disease, however, increased Mirrors in other regions (Coleman et al., 1992). No investigation was at GAP-43 in body fluids of patients with dementia disorders carried out.

A another important structural change in brains of AD patients is synapse loss. In fact, revealed younger Investigations concerning the measurement of the amount of balls, plaques and loss of synapse, that the synapse loss mainly with the extent of Dementia is correlated (Terry et al., 1991). In the latter investigation a reduction in synaptophysin immunoreactivity was observed. A similar Reduction was for other synaptic proteins documented: synaptotagmin, Rab3a, synaptobrevin and Syntaxin (Blennow et al., 1996; Davidsson et al., 1996; Shimohama et al., 1997; Ferrer et al., 1998). There are also several Forms of Parkinson's disease strong signs that synapse proteins play a pathological role. Two mutations in α-synuclein were detected in two rare forms of familial Parkinson's disease, and α-synuclein was characterized as a major component in Lewy bodies. The Lewy body formation in vivo may be from synuclein accumulation resulting in a reduction in fast axon transport or overexpression of synuclein (Jensen et al., 1998). Because the synapse proteins one of the main reasons for the Extent of Dementia is (Terry et al., 1991), it is suitable detection method and quantification of synaptic proteins in body fluids. The presence and the quantification of synapse proteins in CSF has been reported so far not completely explored. Chromogranin has already been used as a marker for synaptic loss used in CSF, however, a reduction was only in 'pure' or Alzheimer's disease Type I (Blennow et al, 1995). Shortly afterwards it was shown synaptotagmin I is present in CSF (Davidsson et al., 1996). In this study, it was shown that synaptotagmin was selective in the left hippocampal formation and Brodmann area 9 of the forehead cortex was reduced by patients with Alzheimer's disease. On the base From CSF pools it was assumed that this reduction was also in the CSF was present but was not quantified. Davidsson et al. (1996) failed to detect Rab3a and synaptophysin in CSF.

Also for the Neurodegeneration caused by the action of certain chemical compounds, Radiation, chemotherapy or hypoxic-ischemic events no accurate diagnostic tools were available. perinatal Asphyxia can be associated with neurological distress. An early and accurate Evaluation of the severity of brain damage after a hypoxic-ischemic Event, however, remains one of the toughest issues with the Neonatal care. So far, clinical electroencephalographic and neurocardiological Assessment, along with cerebral bloodstream studies, the most readily available method. As it became more and more obvious that the modified brain metabolism is due to changes the components are reflected in CSF, the capture of the neurological CSF marker clinical data in the evaluation of hypoxic ischemic Complement events (Garcia-Alix et al., 1994). The connection between neurological CSF markers and behavioral changes in a later one Age after chemotherapy, radiation or a hypoxic event was never determined.

TASKS OF INVENTION

The The object of the present invention is the provision of methods for more specific detection, quantification and / or differential diagnosis of neurodegeneration in an individual.

A Another object of the present invention is the provision a method for more specific detection, quantification and / or for the differential diagnosis of Alzheimer's disease in an individual.

A Another object of the present invention is the provision a method for more specific detection, quantification and / or for the differential diagnosis of Lewy body disease in an individual.

A Another object of the present invention is the provision a method for more specific detection, quantification and / or for the differential diagnosis of Parkinson's disease in an individual.

A Another object of the present invention is the provision a method for more specific detection, quantification and / or for the differential diagnosis of dementia of the frontal lobe / temporalis in an individual.

A Another object of the present invention is the provision of a method for distinguishing Alzheimer's disease from Parkinson's disease.

A Another object of the present invention is the provision of a method for distinguishing Alzheimer's disease from Lewy body dementia.

A Another object of the present invention is the provision a method for specific detection or quantification of vascular Problems with Alzheimer's disease and differential diagnosis various forms of Alzheimer's disease.

A Another object of the present invention is the provision a procedure for diagnosis by chemo or by Exposure to chemical compounds or radiation Neurodegeneration.

A Another object of the present invention is the provision a method of diagnosis of chemotherapy induced Neurodegeneration in on leukemia or brain tumor treated individuals.

A Another object of the present description is the provision a new method for detecting the synapse protein Rab3a in Cerebrospinal fluid.

A Another object of the present description is the provision a new method for the detection of Rab3a in cerebrospinal fluid, that a more specific proof, a more specific quantification and / or differential diagnosis of neurodegeneration in an individual allows.

A Another object of the present description is the provision a new method for the detection of Rab3a in cerebrospinal fluid, that a more specific proof, a more specific quantification and / or differential diagnosis of Alzheimer's disease.

A Another object of the present description is the provision of a new method for detecting the synapse protein α-synuclein in cerebrospinal fluid.

A Another object of the present description is the provision a new method for the detection of α-synuclein in cerebrospinal fluid, that a more specific proof, a more specific quantification and / or differential diagnosis of neurodegeneration in an individual allows.

A Another object of the present description is the provision a new method for the detection of α-synuclein in cerebrospinal fluid, that is a more specific proof or a more specific quantification Alzheimer's disease and / or Lewy body disease, and / or the differential diagnosis of Alzheimer's disease versus Lewy body disease allows.

A Another object of the present invention is the provision a diagnostic kit to carry out a method as described above.

A Another object of the present invention is the provision a method of therapeutic monitoring and / or determination the effectiveness of a particular treatment.

INCOMING DESCRIPTION OF THE INVENTION

The present invention relates to methods for the specific detection, quantification and / or differential diagnosis of neurodegeneration in an individual. These methods involve the determination of the level of at least three neurological markers in one or more body fluids of the individual, thereby reflecting the type and extent of neurodegeneration by a quantitative change in the level of all neurological markers as compared to a control.

Out the examples of the invention shows that by using at least three neurological Mark a specific and sensitive proof of a series from neurodegenerative disorders. It is also apparent do it by using at least three neurological markers allows was in between a series of neurodegenerative conditions distinguished on the basis of clinical diagnosis indistinguishable would.

The Neurodegeneration used in the present application and neurodegenerative state are equivalent and become within the application used interchangeably. These terms include each Suffering of the brain, which is associated with a neuronal dysfunction. Various diseases associated with neurodegeneration, are cited in Wilson et al., (1991). They include Alzheimer's disease, stroke (focal brain injury), diffuse brain injury, Parkinson's disease, Lewy Body Disease, Creutzfeld-Jacob Disease, Dementia of the frontal lobe / temporalis, Guilain Barre syndrome, multiple sclerosis, Normal pressure hydrocephalus, amyotrophic lateral sclerosis, schizophrenia, Depression, neurolathyrisme, epilepsy and asphyxia. This list is but not completely. Other diseases known to be associated with a neuronal dysfunction are also included. Neurodegeneration also includes any type of brain damage or condition of the brain, which is associated with a neuronal malfunction, and which is caused by a specific inducing agent. at a preferred embodiment In the present invention, the specific to be detected, to quantifying and / or differential diagnosing condition selected from the group consisting of Alzheimer's disease, Lewy body disease, Parkinson's disease and dementia of the frontal lobe / temporalis. The Lewy body disease is for Any disease uses the Lewy body in the brainstem, basal forebrain, hypothalamic nucleus and / or neocortex having. The Lewy body disease includes Parkinson's disease, multiple system atrophy and Lewy body dementia.

at a further preferred embodiment In the present invention, the specific to be detected, to quantifying and / or differential-diagnosing neurodegenerative Condition due to hypoxic-ischemic Events, chemotherapy, radiotherapy or exposure to induced chemical compounds or radiation. Neurodegeneration May in particular by chemo or radiotherapy during the Treatment of leukemia or brain tumor can be induced.

The List of neurodegenerative conditions is not complete. Other Conditions, which is a malfunction of the brain, are also added.

Of the Expression "more specific Evidence of neurodegeneration "like it is used in the present invention means that a higher one Sensitivity and specificity for the Assignment of a specific disease or cause a neurological disorder to a specific neurodegenerative condition is obtained when less than three neurological markers can be used for diagnosis.

Of the Expression "Quantification Neurodegeneration ", as used in the present invention means that the degree of neuronal dysfunction due to a particular neurodegenerative Condition is determined The term "differential diagnosis of neurodegeneration" as used in the present The invention relates to the distinction between neurodegenerative states in a way that a certain illness or a particular Cause for a neurological disorder associated with a particular neurodegenerative condition.

• – Inkontaktbringen von einer oder mehreren Körperflüssigkeitsproben, die von einem Individuum erhalten wurden, mit mindestens drei verschiedenen Antikörpern (Primärantikörpern oder Fängerantikörpern), die jeweils einen anderen Marker in den Körperflüssigkeitsproben unter Bedingungen erkennen, die sich zur Erzeugung eines Antigen-Antikörper-Komplexes eignen; und
• – Nachweis der immunologischen Bindung der Antikörper an die Körperflüssigkeitsproben;
• – Rückschluss auf den Spiegel der neurologischen Marker in den Körperflüssigkeiten, wobei sich die Art und das Ausmaß der Neurodegeneration in den quantitativen Änderungen in dem Spiegel sämtlicher neurologischer Marker verglichen mit den Kontrollproben wiederspiegeln.

The specific detection, quantification and / or differential diagnosis of neurodegeneration in an individual is accomplished by the detection of at least three different neurological markers in one of several body fluid samples in the individual; wherein an immunoassay is used, comprising the steps of:

• - contacting one or more body fluid samples obtained from an individual with at least three different antibodies (primary antibodies or capture antibodies) each detecting a different marker in the body fluid samples under conditions suitable for generating an antigen-antibody complex; and
• - proof of immunological binding of the antibodies to the body fluid samples;
• - Conclusion on the level of neurological markers in body fluids, with the nature and extent of neurodegeneration reflected in the quantitative changes in the level of all neurological markers compared to the control samples.

• a) einem Sekundärantikörper (oder Nachweisantikörper) – der ein monoklonaler Antikörper sein kann, der ein spezifisches Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, oder – der ein polyklonaler Antikörper sein kann, der ein spezifisches Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisierter neurologischer Marker oder dem Komplex aus neurologischem Marker und Primärantikörper gereinigt wird.
• b) einem Marker entweder zum spezifischen Tagging oder Kuppeln an den Sekundärantikörper, wobei der Marker ein beliebiger möglicher Marker ist, der dem Fachmann bekannt ist;
• c) geeigneten Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und den Körperflüssigkeitsproben, zwischen dem Sekundärantikörper und dem Komplex aus dem neurologischen Marker und dem Primärantikörper und/oder dem gebundenen Sekundärantikörper und dem Marker andererseits; und
• d) ebenfalls möglicherweise für Standardisierungszwecke gereinigte Proteine oder synthetische Peptide, die mit den Antikörpern reagieren, die zum Nachweisen der neurologischen Marker dienen.

The method of detecting immunological binding can then be carried out by bringing together the antigen-antibody complex formed by the antigen and the antibody which recognizes one of the neurological markers with:

• a) a secondary antibody (or detection antibody) - which may be a monoclonal antibody capable of recognizing a specific epitope of the antigen-antibody complex but not recognizing the primary antibody alone, or - which may be a polyclonal antibody containing a specific epitope of the antigen-antibody complex, but can not recognize the primary antibody alone, the polyclonal antibody preferably being purified by immunoaffinity chromatography using immobilized neurological markers or the neurological marker-primary antibody complex.
• b) a marker either for specific tagging or coupling to the secondary antibody, which marker is any potential marker known to those skilled in the art;
• c) suitable buffer solutions for carrying out the immunological reaction between the antibodies and the body fluid samples, between the secondary antibody and the complex of the neurological marker and the primary antibody and / or the bound secondary antibody and the marker on the other hand; and
• d) also possibly for standardization purposes purified proteins or synthetic peptides that react with the antibodies that serve to detect the neurological markers.

advantageously, are the inventively used antibody in an immobilized state on a suitable carrier. The antibody can up to three (or more in the detection of more than 3 neurological Markers) of different carriers or on the same carrier be present. Are the antibodies on the same carrier (for example in the well of a microtiter plate) the immunological binding of each by a specific Marker can be detected. Alternatively, the antibodies may be on certain places on the same carrier. In the latter In this case, the detection can be done with a general marker, which detects the immunological binding of all these antibodies. The secondary antibody itself bears advantageously a marker or a group for direct or indirect coupling with a marker. The inventive method Alternatively, it can also be put into practice by using any other immunoassay format that suits the Is known in the art.

Of the Term "epitope" refers to those Part of the antigen-antibody complex, specific to an antibody-combining Job is tied. The epitopes can by any techniques of the prior art or can be determined by a variety of Computer prediction models of the prior art predicted become.

The Terms "detecting," "reacting with," "immunological binding," or "generating an antigen-antibody complex," as used in the invention are interpreted to be the bond between the antigen and the antibody takes place under all conditions that have the immunological properties of the antibody and the antigen.

Of the Term body fluids affects all Liquids, those in the human body but not limited to blood, lymph, urine and cerebrospinal fluid (CSF).

In a specific embodiment The present invention relates to a method as described above. taking the body fluid sample selected is from the group consisting of a cerebrospinal fluid sample and a blood test. The blood sample may comprise the entire sample, as it is taken from the patient. The blood sample more preferably comprises a plasma sample or a serum sample.

at the inventive method it is also possible the same markers in two different body fluids (in combination with the detection of at least one other marker) or to detect the same marker in three different body fluids. It For example, two neurological markers in the cerebrospinal fluid and one of these neurological markers is also detected Plasma detected. As shown in the example section, leads as well the detection of the same marker in two different body fluids to a more specific and sensitive detection and to a better differential diagnosis of neurodegeneration compared with the detection of this marker only in a body fluid.

The neurological markers detected in the method of the invention may be any protein associated with particular types of neuronal cells or cell function in which the level in one or more body fluids under conditions of neurodegeneration indicates the course of the disease or the cause of the neurological disorder. Some neurological markers are elevated in one or more body fluids under a particular neurological condition and others are reduced. Any combination of 3, 4, 5, 6, 7, 8 or more neurological markers that have altered levels in a particular body fluid and neurological condition may be used for the specific detection, quantification and / or differential diagnosis of the neurological condition to be used in an individual. Possible neurological markers used for specific detection, quantification and / or differential diagnosis of neurodegeneration include: tau, neuron-specific enolase (NSE), beta-amyloid _(1-42) , beta-amyloid _(1-40) neuromodulin, Synaptic proteins (such as Rab3a, SNAP25, α-synuclein, synapsin, synaptogamin, synaptobrevin, syntaxin, rabphilin, n-sec, cysteine filament protein and others), glial fibrillary acidic protein (GFAP), 5100, IL6, TNF, IL1, IL2, neurofilament ( NF), myelin basic protein (MBP) and 14-3-3. This list is not complete. Other neurological markers that indicate a particular disease process or the cause of a neurological disorder may also be used. The behavior of some of these neurological markers in body fluids of patients suffering from various neurological diseases and brain damage are shown in Table 2.

Any monoclonal or polyclonal antibody produced or present in the art that specifically recognizes any of the above neurological markers may be used to detect the neurological marker. Antibodies that specifically recognize tau include Alz50 (Ghanbari et al., 1990), Ab423 (Harrington et al., 1991), AT8 (International application, published under WO 93/08302), AT120 (Vandermeeren et al., 1993). ; AT180 and AT270 (International Application, published under WO 95/17429) and AT100 (International Application, published under WO 96/04309). However, other prior art antibodies that specifically recognize tau may also be used. Antibodies that specifically recognize NSE include 10Cl and 2E7 and others from Innogenetics (Gent, Belgium), commercially available antibodies obtained from Dako (Glostrup, Denmark, Cat # BBS / NC / VI-H14), from Biogenex (San Ramon, CA, U.S.A., MA055-5C and AM055-5M), from RDI (Flanders, NJ, U.S.A., RDI-TRK4N6), from Roche Diagnostic Systems (Basel, Switzerland; Cat. No. 07 34373), Immunosource (Brussels, Belgium, Cat # CLA 73/5 and CR7041M) and Cortex Biochem (San Leandro, Calif., Cat # CR7047). However, this list of antibodies that recognize NSE is not exhaustive, and other antibodies that are commercially available or described in the art can also be used. Antibodies that specifically recognize β-amyloid include 2H3, 8E5 (Johnson-Wood et al., 1997), 10H3 (Majocha et al., 1992, Friedland et al., 1994), 2G3 (Citron et al., 1996). BA-27 and BC-05 (Suzuki et al., 1994), BNT77 (Asami-Odaka et al., 1995), 3692B (Koenig et al., 1996) 2C11 (Lannfelt et al., 1995), 6E10 (FIG. Kim et al., 1990) and AMY-33 (Stern et al., 1990). However, any other prior art antibodies that specifically recognize β-amyloid can also be used. Antibodies that specifically recognize neuromodulin include NM2 (Oestreicher et al., 1994), NM4 (Six et al., 1992), NM1, NM3, NM6, NM7, and NM8 (Mercken et al., 1992a). However, any other prior art antibody that specifically recognizes neuromodulin may also be used. Antibodies that specifically recognize Rab3a include commercially available antibodies, such as may be obtained from Transduction Labs (Lexington, KY, U.S.A., No. R35520). Also, other antibodies commercially available or described in the art which recognize Rab3a can be used. Antibodies that specifically recognize SNAP25 include commercially available antibodies, such as those available from Serotec (Oxford, UK; Cat No SP12), from Sternberger Monoclonals Inc. (distributed by Affinity Research Products Lim., Mamhead, Exeter, UK Cat # SMI-81), from Chemicon (Temecula, CA, USA, Cat # MAB331), and from Transduction Labs (Lexington, KY, USA; Cat # 535020). This list of antibodies recognizing SNAP25 is not exhaustive and other commercially available or described antibodies recognizing SNAP25 can also be used. Antibodies that specifically recognize an α-synuclein include commercially available antibodies, such as are available from Transduction Labs (Lexington, KY, U.S.A., cat # 563320). Other art-recognized or described antibodies recognizing α-synuclein may also be used. For the specific detection of other synapse proteins which may possibly be used as neurological markers, various antibodies are commercially available and / or known in the art. Antibodies specifically recognizing 5100 include commercially available antibodies as described by Biogenex (San Ramon, Calif., CA, MA058-C and AM058-5M) and Innogenetics (Gent, Belgium, cat. M-011) are available. This list of antibodies recognizing 5100 is not exhaustive, and other antibodies commercially available or described in the art which recognize S100 may also be used. Antibodies specifically recognizing 14-3-3 include commercially available antibodies as described by Santa Cruz Biotechnology (Santa Cruz, Calif., U.S.A., sc-1657) and Transduction Labs (Lexington, Ky., USA; Cat No. F46820) are available. This list of antibodies recognizing 14-3-3 is not exhaustive and others commercially available in the art antibodies recognizing 14-3-3 can also be used. Antibodies that specifically recognize neurofilament include commercially available antibodies such as those available from Innogenetics (Ghent, Belgium, Cat No M-011 and M-005) and from Alexis (Läufelingen, Switzerland; Cat. No. BC-4000 -A-L001 and BC-4010-A-L001) are available. This list of antibodies recognizing neurofilament is not exhaustive, and other antibodies commercially available or described in the art which recognize neurofilament may also be used.

Also, fragments derived from these monoclonal antibodies, such as Fab, F (ab) ' ₂ , ssFv ("variable single chain fragment"), and other antibody-like constructs which retain the variable region of the antibody, provided they have retained the original binding properties be used in a method according to the invention. Such fragments are usually generated by, for example, enzymatic cleavage of the antibodies with papain, pepsin or other proteases. The person skilled in the art knows that monoclonal antibodies or their fragments can be modified for various applications. Also, miniantibodies and polyvalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptobodies can be used in a method of the invention. The preparation and use of these fragments and polyvalent antibodies has been extensively described in International Patent Application WO 98/29442.

The in a method according to the invention used antibodies can be humanized versions of mouse monoclonal antibodies, prepared by DNR recombinant technology using mouse and / or human genomic DNA sequences, encoding the H and L chains, or cDNA clones, encode the H and L chains. The term "humanized antibody" means that at least a portion the framework regions of an immunoglobulin of human immunoglobulin sequences is derived. The antibodies used in a method according to the invention can by a suitable label of the enzymatic, fluorescent or radioactive type are marked.

• – Tau, β-Amyloid_(1-42) und Neuromodulin; oder
• – Tau, neuronenspezifische Enolase und Neuromodulin oder
• – Tau, Phospho-Tau, β-Amyloid_(1-42);

In a specific embodiment of the invention, at least one of the neurological markers to be detected in a method as described above is selected from the group consisting of tau, phospho-tau, (β-amyloid _(1-42) , β-amyloid _(1-40) , neuromodulin, neuron-specific enolase and / or synapse proteins One possible combination of 3, 4, 5, 6, 7, 8, or more markers, one of which is selected from the above group, may be used for specific detection It is also clear that more than one (ie 2, 3, 4, 5, 6, 7, or more) or even all the neurological markers present in In a more specific embodiment of the invention, one, more preferably two, most preferably three of the neurological Mar ker, which are to be detected in a method according to the invention as described above, selected from the following groups:

• Tau, β-amyloid _(1-42) and neuromodulin; or
• - Tau, neuron-specific enolase and neuromodulin or
• Tau, phospho-tau, β-amyloid _(1-42) ;

In another more specific embodiment, tau is detected in a body fluid, preferably CSF, and β-amyloid _(1-42) is detected in two different body fluids, preferably CSF and plasma.

at Another more specific embodiment is at least one the biological marker used in a procedure as described above to be detected, a synapse protein selected from the Group consisting of Rab3a, SNAP25 and α-synuclein. Any combination of 3, 4, 5, 6, 7, 8 or more markers, one of which group of synapse proteins is selected for the specific group Detection, quantification and / or differential diagnosis of neurodegeneration in an individual.

• – Inkontaktbringen von einer Zerebrospinalflüssigkeitsprobe, die von einem Individuum erhalten wurde, mit einem monoklonalen Antikörper (Primärantikörper oder Fängerantikörper), der Rab3a unter Bedingungen erkennt, die sich zur Erzeugung eines Antigen-Antikörper-Komplexes eignen; und
• – Nachweisen der immunologischen Bindung des Antikörpers an die Zerebrospinalflüssigkeitsprobe.

Accordingly, the present description relates to a novel method for the detection of Rab3a in cerebrospinal fluid comprising at least the following steps:

• Contacting a cerebrospinal fluid sample obtained from an individual with a monoclonal antibody (primary antibody or capture antibody) that recognizes Rab3a under conditions suitable for generating an antigen-antibody complex; and
• - Detecting the immunological binding of the antibody to the cerebrospinal fluid sample.

For the detection of Rab3a in brain tissue, although antibodies are specifically Rab3a he available However, the detection of Rab3a in cerebrospinal fluid was not so obvious. By their method used Davidsson et al. (1996) did not detect Rab3a in the cerebrospinal fluid. Any antibody that allows specific detection of Rab3a in the cerebrospinal fluid can be used in this new procedure. A preferred monoclonal antibody for use in the method of the invention can be obtained from Transduction Labs (Lexington, KY, U.S.A., R35520).

Of the used according to the invention monoclonal antibodies is advantageously in an immobilized state on one suitable carrier. The inventive method Alternatively, it can be put into practice by using any another immunoassay format is used, known to those skilled in the art is.

• a) einem Sekundärantikörper (oder Nachweisantikörper) – der ein monoklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, oder – der ein polyklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem Rab3a oder dem Komplex aus Rab3a und Primärantikörper gereinigt wird.
• b) einem Marker entweder zum spezifischen Tagging oder Kuppeln an den Sekundärantikörper, wobei der Marker ein beliebiger möglicher Marker ist, der dem Fachmann bekannt ist;
• c) geeigneten Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe zwischen dem Sekundärantikörper und dem Komplex aus dem neurologischen Marker und dem Primärantikörper und/oder dem gebundenen Sekundärantikörper und dem Marker; und
• d) ebenfalls möglicherweise für Standardisierungszwecke gereinigten Proteinen oder synthetischen Peptiden, die mit den Antikörpern reagieren, die Rab3a erkennen.

The method for detecting the immunological binding can then be carried out by bringing together the antigen-antibody complex formed by the antigen and the antibody recognizing Rab3a with:

• a) a secondary antibody (or detection antibody) - which may be a monoclonal antibody that can recognize an epitope of the antigen-antibody complex but can not recognize the primary antibody alone, or - which may be a polyclonal antibody that is an epitope of the antigen Antibody complex but can not recognize the primary antibody alone, the polyclonal antibody preferably being purified by immunoaffinity chromatography using immobilized Rab3a or the complex of Rab3a and primary antibody.
• b) a marker either for specific tagging or coupling to the secondary antibody, which marker is any potential marker known to those skilled in the art;
• c) suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample between the secondary antibody and the complex of the neurological marker and the primary antibody and / or the bound secondary antibody and the marker; and
• d) also possibly for standardization purposes purified proteins or synthetic peptides that react with the antibodies that recognize Rab3a.

As in the examples of the invention Illustratively, a polyclonal Rab3a serum can be used as the detection antibody become.

Of the Secondary antibody itself bears advantageously a marker or group for direct or indirect coupling with a marker.

• – Inkontaktbringen von einer Zerebrospinalflüssigkeitsprobe, die von einem Individuum erhalten wurde, mit einem monoklonalen Antikörper (Primärantikörper oder Fängerantikörper), der SNAP25 unter Bedingungen erkennt, die sich zur Erzeugung eines Antigen-Antikörper-Komplexes eignen; und
• – Nachweisen der immunologischen Bindung des Antikörpers an die Zerebrospinalflüssigkeitsprobe.

The present description also relates to a novel method for detecting SNAP25 in cerebacterial fluid comprising at least the following steps:

• Contacting a cerebrospinal fluid sample obtained from an individual with a monoclonal antibody (primary antibody or capture antibody) which recognizes SNAP25 under conditions suitable for the production of an antigen-antibody complex; and
• - Detecting the immunological binding of the antibody to the cerebrospinal fluid sample.

For the proof Although SNAP25 in brain tissue has antibodies that specifically recognize SNAP25, however, the detection of SNAP25 was in cerebrospinal fluid not so obvious and has not been shown before.

Everyone Antibody, specific detection of SNAP25 in the cerebrospinal fluid allows can be used in this new process. A preferred one monoclonal antibody for use in the method of the invention may be obtained from Serotec (Oxford UK, cat # SP12) from Sternberger Monoclonals Inc. (Distrib by Affinity Research Products Lim, Mamhead, Exeter, UK; Cat SMI-81) from Chemicon (Temecula, CA, USA, Cat.No. MAB 331) or from Transduction Labs (Lexington, KY, USA; Cat # 535020).

Of the used according to the invention monoclonal antibodies is advantageously in an immobilized state on one suitable carrier, The inventive method Alternatively, it can be put into practice by using any another immunoassay format is used, known to those skilled in the art is.

• a) einem Sekundärantikörper (oder Nachweisantikörper) – der ein monoklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, oder – der ein polyklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem SNAP25 oder dem Komplex aus SNAP25 und Primärantikörper gereinigt wird.
• b) einem Marker entweder zum spezifischen Tagging oder Kuppeln an den Sekundärantikörper, wobei der Marker ein beliebiger möglicher Marker ist, der dem Fachmann bekannt ist;
• c) geeigneten Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe, zwischen dem Sekundärantikörper und dem Komplex aus dem neurologischen Marker und dem Primärantikörper und/oder dem gebundenen Sekundärantikörper und dem Marker; und
• d) ebenfalls möglicherweise für Standardisierungszwecke gereinigten Proteinen oder synthetischen Peptiden, die mit den Antikörpern reagieren, die SNAP25 erkennen.

The method for detecting the immunological binding can then be carried out by bringing together the antigen-antibody complex formed by the antigen and the antibody which recognizes SNAP25 with:

• a) a secondary antibody (or detection antibody) - which may be a monoclonal antibody that can recognize an epitope of the antigen-antibody complex but can not recognize the primary antibody alone, or - which may be a polyclonal antibody that is an epitope of the antigen Antibody complex but can not recognize the primary antibody alone, the polyclonal antibody preferably being purified by immunoaffinity chromatography using immobilized SNAP25 or the complex of SNAP25 and primary antibody.
• b) a marker either for specific tagging or coupling to the secondary antibody, which marker is any potential marker known to those skilled in the art;
• c) suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the complex of the neurological marker and the primary antibody and / or the bound secondary antibody and the marker; and
• d) also possibly for standardization purposes purified proteins or synthetic peptides that react with the antibodies that recognize SNAP25.

As in the examples of the invention Illustratively, a polyclonal SNAP25 serum can be used as the detection antibody become.

Of the Secondary antibody itself wears advantageously a marker or group for direct or indirect coupling with a marker.

• – Inkontaktbringen von einer Zerebrospinalflüssigkeitsprobe, die von einem Individuum erhalten wurde, mit einem monoklonalen Antikörper (Primärantikörper oder Fängerantikörper), der α-Synuclein unter Bedingungen erkennt, die sich zur Erzeugung eines Antigen-Antikörper-Komplexes eignen; und
• – Nachweisen der immunologischen Bindung des Antikörpers an die Zerebrospinalflüssigkeitsprobe.

The present description also relates to a novel method for detecting α-synuclein in cerebrospinal fluid comprising at least the following steps:

• - contacting a cerebrospinal fluid sample obtained from an individual with a monoclonal antibody (primary antibody or capture antibody) that recognizes α-synuclein under conditions suitable for generating an antigen-antibody complex; and
• - Detecting the immunological binding of the antibody to the cerebrospinal fluid sample.

For the proof of α-synuclein There are antibodies in brain tissue available, the specific α-synuclein however, detection of SNAP25 was in cerebrospinal fluid not so obvious. Because the presence of α-synuclein in cerebrospinal fluid it was even doubtful whether α-synuclein was ever present in CSF is available. First, the inventors were able to show that α-synuclein in cerebrospinal fluid is available. In addition, an accurate method for quantitative detection has been developed of α-synuclein in cerebrospinal fluid developed. The inventors also showed that α-synuclein under certain neurodegenerative conditions is changed, including, but not limited to Lewy body disease.

Everyone Antibody, the specific detection of α-synuclein in the cerebrospinal fluid allows can be used in this new process. A preferred one monoclonal antibody For use in the method of the invention, Transduction Labs (Lexington, KY, USA; Cat # R35520).

Of the used according to the invention monoclonal antibodies is advantageously in an immobilized state on one suitable carrier. This immobilized state may be a microtiter plate containing coated anti-IgG is or not. The inventive method may alternatively put into practice by any other immunoassay format is used, which is known in the art.

• a) einem Sekundärantikörper (oder Nachweisantikörper) – der ein monoklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, oder – der ein polyklonaler Antikörper sein kann, der ein Epitop des Antigen-Antikörper-Komplexes erkennen kann, aber nicht den Primärantikörper allein erkennen kann, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem α-Synuclein oder dem Komplex aus α-Synuclein und Primärantikörper gereinigt wird.
• b) einem Marker entweder zum spezifischen Tagging oder Kuppeln an den Sekundärantikörper, wobei der Marker ein beliebiger möglicher Marker ist, der dem Fachmann bekannt ist;
• c) geeigneten Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe, zwischen dem Sekundärantikörper und dem Komplex aus dem neurologischen Marker und dem Primärantikörper und/oder dem gebundenen Sekundärantikörper und dem Marker; und
• d) ebenfalls möglicherweise für Standardisierungszwecke gereinigten Proteinen oder synthetischen Peptiden, die mit den Antikörpern reagieren, die α-Synuclein erkennen.

The method for detecting the immunological binding can then be carried out by bringing together the antigen-antibody complex formed by the antigen and the antibody which recognizes α-synuclein:

• a) a secondary antibody (or detection antibody) - which may be a monoclonal antibody that can recognize an epitope of the antigen-antibody complex but can not recognize the primary antibody alone, or - which may be a polyclonal antibody that is an epitope of the antigen Antibody complex, but can not recognize the primary antibody alone, with the polyclonal antibody preferably purified by immunoaffinity chromatography using immobilized α-synuclein or the complex of α-synuclein and primary antibody.
• b) a marker either for specific tagging or coupling to the secondary antibody, which marker is any potential marker known to those skilled in the art;
• c) suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the complex of the neurological marker and the primary antibody and / or the bound secondary antibody and the marker; and
• d) also possibly for standardization purposes purified proteins or synthetic peptides that react with the antibodies that recognize α-synuclein.

Of the Secondary antibody itself wears advantageously a marker or group for direct or indirect coupling with a marker.

at a preferred embodiment can these methods for the determination of Rab3a, SNAP25 and / or α-synuclein in combination with a method of detecting one or more neurological Markers for specific verification, quantification and / or for Differential diagnosis of neurodegeneration used in an individual become.

In a still more preferred embodiment, these methods can be used to detect Rab3a, SNAP25 and / or α-synuclein in combination with a method of detecting one or more neurological markers selected from the group consisting of tau, phospho-tau, β-amyloid _(1-42) , β-amyloid _(1-42) , neuromodulin, neuron-specific enolase (NSE).

• – Tau, Phospho-Tau, NSE, β-Amyloid_(1-42), β-Amyloid_(1-40) Neuromodulin oder Rab3a; oder
• – Tau, Phospho-Tau, NSE, β-Amyloid_(1-42), β-Amyloid(1-40) Neuromodulin oder SNAP25; oder
• – Tau, Phospho-Tau, NSE, β-Amyloid_(1-42), β-Amyloid(1-40), Neuromodulin oder α-Synuclein.

In particular, the neurological markers used for the specific detection, quantification and / or differential diagnosis of neurodegeneration may be selected from one of the following groups:

• Tau, phospho-tau, NSE, β-amyloid _(1-42) , β-amyloid _(1-40) neuromodulin or Rab3a; or
• Tau, phospho-tau, NSE, β-amyloid _(1-42) , β-amyloid (1-40) neuromodulin or SNAP25; or
• Tau, phospho-tau, NSE, β-amyloid _(1-42) , β-amyloid (1-40), neuromodulin or α-synuclein.

each possible Combination of 3, 4, 5, 6 or 7 markers from the above groups can be used for specific detection, quantification and / or differential diagnosis of neurodegeneration in an individual.

at another embodiment can the methods for detecting Rab3a, SNAP25 and / or α-synuclein together for specific detection, quantification and / or differential diagnosis used by neurodegeneration. The present invention thus relates to a method wherein two or three of the neurological Marker selected are selected from the group consisting of Rab3a, SNAP35 and α-synuclein.

• – zumindest der Spiegel von α-Synuclein in einer Zerebrospinalflüssigkeitsprobe bestimmt wird; und/oder
• – der Spiegel von Tau, β-Amyloid_(1-42), und α-Synuclein in einer Zerebrospinalflüssigkeitsprobe bestimmt wird.

A very specific embodiment relates to a method as described above for the specific detection or quantification of Alzheimer's disease and / or Lewy body disease and / or for the differential diagnosis of Alzheimer's disease versus Lewy body disease, wherein

• At least the level of α-synuclein in a cerebrospinal fluid sample is determined; and or
• - the level of tau, β-amyloid _(1-42) , and α-synuclein in a cerebrospinal fluid sample is determined.

• – der Spiegel von Tau, β-Amyloid_(1-42), und Rab3a in einer Zerebrospinalflüssigkeitsprobe bestimmt wird; oder
• – der Spiegel von Tau, β-Amyloid_(1-42), und SNAP25 in einer Zerebrospinalflüssigkeitsprobe bestimmt wird. Eine weitere sehr spezifische Ausführungsform betrifft ein Verfahren zum spezifischen Nachweis oder zur Quantifizierung von Alzheimer-Krankheit und/oder Parkinson-Krankheit und/oder zur Differenzialdiagnose von Alzheimer-Krankheit gegenüber Parkinson-Krankheit, wobei der Spiegel von Tau, β-Amyloid_(1-42), und Neuromodulin in einer Zerebrospinalflüssigkeitsprobe bestimmt wird.

Another very specific embodiment relates to a method for specifically detecting or quantifying Alzheimer's disease and / or for differential diagnosis of Alzheimer's disease versus another dementia, wherein:

• - the level of tau, β-amyloid _(1-42) , and Rab3a in a cerebrospinal fluid sample is determined; or
• - the level of tau, β-amyloid _(1-42) , and SNAP25 in a cerebrospinal fluid sample is determined. Another very specific embodiment relates to a method for the specific detection or quantification of Alzheimer's disease and / or Parkinson's disease and / or for the differential diagnosis of Alzheimer's disease against Parkinson's disease, wherein the level of tau, β-amyloid _{(1- 42)} , and neuromodulin is determined in a cerebrospinal fluid sample.

Another very specific embodiment relates to a method for specific detection or for quantifying neurodegeneration induced by chemotherapy, exposure to chemical compounds and / or radiation, wherein the level of tau, neuron-specific enolase and neuromodulin in a cerebrospinal fluid sample is determined.

A another very specific embodiment relates to a method for specific detection or quantification of neurodegeneration induced by chemotherapy, exposure of chemical compounds and / or irradiation, in an individual, that on leukemia or brain tumor is treated, wherein the level of tau, neuron-specific Enolase and neuromodulin in a cerebrospinal fluid sample is determined.

A other very specific embodiment relates to a method for specific detection or quantification neurodegeneration induced by prenatal asphyxia, wherein at least three neurological markers are detected.

Another very specific embodiment relates to a method for specifically detecting or quantifying the dementia of the frontal lobe / temporalis and / or for differential diagnosis of dementia of the frontal lobe / temporal lobe against another dementia in which the level of tau, phospho-tau and β Amyloid _{(1-42) is determined} in a cerebrospinal fluid sample.

• – Tau und β-Amyloid_(1-42) quantitativ in einer Zerebrospinalflüssigkeitsprobe bestimmt wird, und der Spiegel von β-Amyloid_(1-42) quantitativ in einer Plasmaprobe bestimmt wird; oder
• – Phospho-Tau und β-Amyloid_(1-42) quantitativ in einer Zerebrospinalflüssigkeitsprobe bestimmt wird und der Spiegel von β-Amyloid_(1-42) quantitativ in einer Plasmaprobe bestimmt wird; oder
• – Tau und Phospho-Tau quantitativ in einer Zerebrospinalflüssigkeitsprobe bestimmt wird, und der Spiegel von β-Amyloid_(1-42) quantitativ in einer Plasmaprobe bestimmt wird; oder
• – Tau, Phospho-Tau und β-Amyloid_(1-42) quantitativ in einer Zerebrospinalflüssigkeitsprobe bestimmt wird.

Another very specific embodiment relates to a method for specifically detecting or quantifying vascular problems in Alzheimer's disease, for differential diagnosis of various forms of Alzheimer's disease, and / or for differential diagnosis of Alzheimer's disease over other dementia, wherein at least the level of:

• - Tau and β-amyloid _{(1-42) are} quantitated in a sample of cerebrospinal fluid, and the level of β-amyloid _{(1-42) is} quantified in a plasma sample; or
• - Phospho-tau and β-amyloid _{(1-42) are} quantitated in a cerebrospinal fluid sample and the level of β-amyloid _{(1-42) is} quantitated in a plasma sample; or
• - Tau and phospho-tau are determined quantitatively in a cerebrospinal fluid sample, and the level of β-amyloid _{(1-42) is determined} quantitatively in a plasma sample; or
• - Tau, phospho-tau and β-amyloid _{(1-42) is} quantitated in a cerebrospinal fluid sample.

The above methods for specific detection, for quantification and / or for the differential diagnosis of neurodegeneration in a Individual by determination of at least three neurological markers in body fluids of the individual, can used alone, in combination with easy to monitor neurological endpoints (eg, white blood cell count) or in Combination with the measurement of drug concentration in plasma.

The The present invention also relates to a diagnostic kit for the specific Detection, quantification and / or differential diagnosis of neurodegeneration in a subject comprising at least three Antibody, each having a different neurological marker in one or more Body fluid samples of the individual.

The The present invention particularly relates to a diagnostic kit for specific detection, quantification and / or differential diagnosis of neurodegeneration in an individual comprising at least one Carrier, like a microtiter plate, together or in separate wells, with at least three antibodies, each having a different neurological marker in one or more Body fluid samples of the individual.

• – einen Träger, wie eine Mikrotiterplatte, umfassend zusammen oder in gesonderten Vertiefungen, mindestens drei Antikörper (Primärantikörper oder Fängerantikörper), die jeweils einen anderen neurologischen Marker erkennen;
• – Sekundärantikörper (Nachweisantikörper), die jeweils einen der Komplexe aus neurologischem Marker und Primärantikörper erkennen: – der ein monoklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus neurologischem Marker und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein; oder – der ein polyklonaler Antikörper sein kann, der einen immunologischen Komplex mit Epitopen des Komplexes aus neurologischem Marker und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem neurologischen Marker oder dem Komplex aus neurologischem Marker und Primärantikörper gereinigt werden kann;
• – möglicherweise einen Marker zum spezifischen Tagging oder Kuppeln mit den Sekundärantikörpern;
• – möglicherweise geeignete Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Primärantikörpern und der Körperflüssigkeitsprobe, zwischen den Sekundärantikörpern und dem Komplex aus neurologischem Marker und Primärantikörper und/oder zwischen. den gebundenen Sekundärantikörpern und dem Marker;
• – möglicherweise für Standardisierungszwecke gereinigte Proteine oder synthetische Peptide, die spezifisch von den Antikörpern des Kits erkannt werden, das zum Nachweis des neurologischen Markers verwendet wird: In spezifischen Ausführungsformen betrifft die vorliegende Erfindung Diagnose-Kits wie oben beschrieben, die jeweils zum Durchführen von einem oder mehreren Verfahren wie oben beschrieben ausgelegt sind. Insbesondere betrifft die vorliegende Erfindung ein Diagnose-Kit wie oben beschrieben, umfassend mindestens Antikörper, die spezifisch erkennen:
• – α-Synuclein oder
• – Tau, β-Amyloid_(1-42) und α-Synuclein; oder
• – Tau, β-Amyloid_(1-42) und Rab3a; oder
• – Tau, β-Amyloid_(1-42), und SNAP25; oder
• – Tau, β-Amyloid_(1-42) und Neuromodulin; oder
• – Tau, neuronenspezifische Enolase und Neuromodulin; oder
• – Tau, Phospho-Tau und β-Amyloid_(1-42); oder
• – Tau und β-Amyloid_(1-42);

The present invention also relates to a kit for the specific detection, quantification and / or differential diagnosis of neurodegeneration in a subject, comprising:

• A carrier, such as a microtiter plate, comprising together or in separate wells, at least three antibodies (primary antibody or capture antibody) each recognizing a different neurological marker;
• - Secondary antibodies (detection antibodies) each recognizing one of the complexes of neurological marker and primary antibody: - which may be a monoclonal antibody capable of forming an immunological complex with an epitope of the complex of neurological marker and primary antibody, but not with the primary antibody alone; or - which may be a polyclonal antibody capable of forming an immunological complex with epitopes of the neurological marker-primary antibody complex, but not the primary antibody alone, wherein the polyclonal antibody is preferably purified by immunoaffinity chromatography using immobilized neurological marker or neurological marker-primary antibody complex;
• Possibly a marker for specific tagging or coupling with the secondary antibodies;
• Possibly suitable buffer solutions for carrying out the immunological reaction between the primary antibodies and the body fluid sample, between the secondary antibodies and the complex of neurological marker and primary antibody and / or between. the bound secondary antibodies and the marker;
• Potentially purified for standardization proteins or synthetic peptides specifically recognized by the antibodies of the kit used to detect the neurological marker: In specific embodiments, the present invention relates to diagnostic kits as described above, each for performing one or more of several methods are designed as described above. In particular, the present invention relates to a diagnostic kit as described above comprising at least antibodies which specifically recognize:
• - α-synuclein or
• Tau, β-amyloid _(1-42) and α-synuclein; or
• Tau, β-amyloid _(1-42) and Rab3a; or
• Tau, β-amyloid _(1-42) , and SNAP25; or
• Tau, β-amyloid _(1-42) and neuromodulin; or
• - Tau, neuron-specific enolase and neuromodulin; or
• Tau, phospho-tau and β-amyloid _(1-42) ; or
• Tau and β-amyloid _(1-42) ;

The present description relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising at least one monoclonal antibody recognizing Rab3a.

The present description also relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising at least one carrier, as a microtiter plate comprising a monoclonal antibody, the Rab3a recognizes.

• – mindestens einen Träger, wie eine Mikrotiterplatte, umfassend einen monoklonalen Antikörper, der Rab3a erkennt (Primärantikörper oder Fängerantikörper);
• – einen Sekundärantikörper (oder Nachweisantikörper): – der ein monoklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus Rab3a und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein; oder – der ein polyklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus Rab3a und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem Rab3a oder dem Komplex aus Rab3a und Primärantikörper gereinigt werden kann;
• – möglicherweise einen Marker zum spezifischen Tagging oder Kuppeln mit dem Sekundärantikörper;
• – möglicherweise geeignete Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe, zwischen dem Sekundärantikörper und dem Komplex aus Rab3a und Primärantikörper und/oder zwischen den gebundenen Sekundärantikörpern und dem Marker;
• – möglicherweise für Standardisierungszwecke gereinigte Proteine oder synthetische Peptide, die spezifisch von den Antikörpern des Kits erkannt werden, das zum Nachweis von Rab3a verwendet wird:

Die vorliegende Beschreibung betrifft auch ein Kit zum Nachweis von SNAP25 in Zerebrospinalflüssigkeit, umfassend mindestens einen monoklonalen Antikörper, der SNAP25 erkennt.In particular, the present description relates to a kit for the detection of Rab3a in cerebrospinal fluid, comprising:

• At least one carrier, such as a microtiter plate, comprising a monoclonal antibody recognizing Rab3a (primary antibody or capture antibody);
• A secondary antibody (or detection antibody): which may be a monoclonal antibody capable of forming an immunological complex with an epitope of the complex of Rab3a and primary antibody, but not with the primary antibody alone; or - which may be a polyclonal antibody capable of forming an immunological complex with an epitope of the complex of Rab3a and primary antibody but not with the primary antibody alone, the polyclonal antibody preferably being purified by immunoaffinity chromatography using immobilized Rab3a or the complex of Rab3a and primary antibodies can be;
• Possibly a marker for specific tagging or coupling with the secondary antibody;
• Possibly suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the complex of Rab3a and primary antibodies and / or between the bound secondary antibodies and the marker;
• - possibly purified for standardization purposes, or synthetic peptides specifically recognized by the antibodies of the kit used to detect Rab3a:

The present specification also relates to a kit for detecting SNAP25 in cerebrospinal fluid comprising at least one monoclonal antibody recognizing SNAP25.

The present description also relates to a kit for the detection of SNAP25 in cerebrospinal fluid, comprising at least one carrier, as a microtiter plate comprising a monoclonal antibody, the SNAP25 detects.

• – mindestens einen Träger, wie eine Mikrotiterplatte, umfassend einen monoklonalen Antikörper, der SNAP25 erkennt (Primärantikörper oder Fängerantikörper);
• – einen Sekundärantikörper (oder Nachweisantikörper): – der ein monoklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus SNAP25 und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein; oder – der ein polyklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus SNAP25 und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem SNAP25 oder dem Komplex aus SNAP25 und Primärantikörper gereinigt werden kann;
• – möglicherweise einen Marker zum spezifischen Tagging oder Kuppeln mit dem Sekundärantikörper;
• – möglicherweise geeignete Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe, zwischen dem Sekundärantikörper und dem Komplex aus SNAP25 und Primärantikörper und/oder zwischen dem gebundenen Sekundärantikörper und dem Marker;
• – möglicherweise für Standardisierungszwecke gereinigte Proteine oder synthetische Peptide, die spezifisch von den Antikörpern des Kits erkannt werden, das zum Nachweis von SNAP25 verwendet wird.

In particular, the present description relates to a kit for the detection of SNAP25 in cerebrospinal fluid, comprising:

• At least one carrier, such as a microtiter plate, comprising a monoclonal antibody, the SNAP25 recognizes (primary antibody or capture antibody);
• A secondary antibody (or detection antibody): which may be a monoclonal antibody capable of forming an immunological complex with an epitope of the complex of SNAP25 and primary antibody, but not with the primary antibody alone; or - which may be a polyclonal antibody capable of forming an immunological complex with an epitope of the SNAP25 complex and primary antibody but not the primary antibody alone, preferably the polyclonal antibody purified by immunoaffinity chromatography using immobilized SNAP25 or the complex of SNAP25 and primary antibody can be;
• Possibly a marker for specific tagging or coupling with the secondary antibody;
• Possibly suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the complex of SNAP25 and primary antibodies and / or between the bound secondary antibody and the marker;
• - possibly purified for standardization proteins or synthetic peptides that are specifically recognized by the antibodies of the kit used to detect SNAP25.

The present description also relates to a kit for the detection of α-synuclein in cerebrospinal fluid, comprising at least one monoclonal antibody recognizing α-synuclein.

The present description also relates to a kit for the detection of α-synuclein in cerebrospinal fluid, comprising at least one carrier, as a microtiter plate comprising a monoclonal antibody, the α-synuclein recognizes.

• – mindestens einen Träger, wie eine Mikrotiterplatte, umfassend einen monoklonalen Antikörper, der α- Synuclein erkennt (Primärantikörper oder Fängerantikörper), der direkt an die Mikrotiterplatte gebunden ist, möglicherweise über einen IgG-Antikörper;
• – einen Sekundärantikörper (oder Nachweisantikörper): – der ein monoklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus α-Synuclein und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein; oder – der ein polyklonaler Antikörper sein kann, der einen immunologischen Komplex mit einem Epitop des Komplexes aus α-Synuclein und Primärantikörper bilden kann, aber nicht mit dem Primärantikörper allein, wobei der polyklonale Antikörper vorzugsweise durch Immunaffinitätschromatographie mittels immobilisiertem α-Synuclein oder dem Komplex aus α-Synuclein und Primärantikörper gereinigt werden kann;
• – möglicherweise einen Marker zum spezifischen Tagging oder Kuppeln mit dem Sekundärantikörper;
• – möglicherweise geeignete Pufferlösungen zum Durchführen der immunologischen Reaktion zwischen den Antikörpern und der Zerebrospinalflüssigkeitsprobe, zwischen dem Sekundärantikörper und dem Komplex aus α-Synuclein und Primärantikörper und/oder zwischen dem gebundenen Sekundärantikörper und dem Marker;
• – möglicherweise für Standardisierungszwecke gereinigte Proteine oder synthetische Peptide, die spezifisch von den Antikörpern des Kits erkannt werden, das zum Nachweis von α-Synuclein verwendet wird.

In particular, the present description relates to a kit for the detection of α-synuclein in cerebrospinal fluid, comprising:

• At least one support, such as a microtiter plate, comprising a monoclonal antibody which recognizes α-synuclein (primary antibody or capture antibody) bound directly to the microtiter plate, possibly via an IgG antibody;
• A secondary antibody (or detection antibody): which may be a monoclonal antibody capable of forming an immunological complex with an epitope of the α-synuclein complex and primary antibody but not with the primary antibody alone; or - which may be a polyclonal antibody capable of forming an immunological complex with an epitope of the α-synuclein complex and primary antibody, but not with the primary antibody alone, preferably the polyclonal antibody by immunoaffinity chromatography using immobilized α-synuclein or the complex α-synuclein and primary antibodies can be purified;
• Possibly a marker for specific tagging or coupling with the secondary antibody;
• Possibly suitable buffer solutions for carrying out the immunological reaction between the antibodies and the cerebrospinal fluid sample, between the secondary antibody and the complex of α-synuclein and primary antibody and / or between the bound secondary antibody and the marker;
• - possibly purified for standardization proteins or synthetic peptides that are specifically recognized by the antibodies of the kit used to detect α-synuclein.

The The present invention also relates to the use of any Method or any kit as described above for therapeutic monitoring and / or to determine the efficacy of a particular treatment.

Tabelle 3. Titer, erhalten nach der Immunisierung von Mäusen mit α- Synuclein.

Tabelle 4. Spiegel der intrazellulären Proteine in Zerebrospinalflüssigkeit (Mittelw. ±SD)

• AU = willkürliche Einheiten.
• * Signifikant unterschiedlich von der Kontrolle (p ≤ 0.05; Whitney test).
• **Daten für β-Amyloid_(1-42)-Spiegel in Zerebrospioalflüssigkeit aus Kontrollpatienten waren nicht verfügbar.

Tabelle 5. Wahrscheinlichkeitsverhältnisse für jeden Marker allein für Markerkombinationen.

• *Daten für die β-Amyloid_(1-42)-Spiegel in Zerebrospinalflüssigkeit aus Kontrollpatienten wurden aus einer anderen Untersuchung erhalten.

Tabelle 8. Durchschnittliche Spiegel in Zerebrospinalflüssigkeit von Tau, β- Amyloid_(1-42) und Neuromodulin für 4 Patientengruppen, wie in Beispiel 7 beschrieben.

• *Signifikant unterschiedlich von Kontrollen (p < 0,01; zweigabeliger Wilcoxon-Test) Die Spiegel sind ausgedrückt als pg/ml, Mittelwert ± Standardabweichung.

The following examples are merely illustrative of the present invention. TABLES Table 1. Neurological complications of chemotherapeutic agents (partial list)

Table 3. Titers obtained after immunization of mice with α-synuclein.

Table 4. Levels of intracellular proteins in cerebrospinal fluid (mean ± SD)

• AU = arbitrary units.
• * Significantly different from control (p ≤ 0.05; Whitney test).
• ** Data for β-amyloid _(1-42) levels in cerebral spinal fluid from control patients were not available.

Table 5. Probability ratios for each marker for marker combinations only.

• * Data for the β-amyloid _(1-42) levels in cerebrospinal fluid from control patients were obtained from another study.

Table 8. Average levels in cerebrospinal fluid of tau, β-amyloid _(1-42) and neuromodulin for 4 patient groups as described in Example 7.

• * Significantly different from controls (p <0.01, bifurcated Wilcoxon test) Levels are expressed as pg / ml, mean ± standard deviation.

FIGURE LEGENDS

1 , Western Blot as described in Example 1.3. Shown is Rab3a immunoreactivity in the temporal cortex of Alzheimer's disease and control brains. 1. 12.8 μl control patient # 3; 2. 6.4 μl of control patient # 3; 3. 3.2 μl control patient # 3; 4. 1.6 μl control patient # 3; 5. 1.0 μl control patient # 3; 6. 10 μl of control patient # 3; 7. 10 μl AD patient # 1; 8. 10 μl AD patient # 2; 9. 10 μl control patient # 1; 10th 10 μl of control patient # 102; 11. 10 μl AD patient No. 3. 12. 10 μl control patient # 4.

2 , Stability of synapse protein Rab3a (a) and SNAP25 (b) in cerebrospinal fluid. The degradation of the synapse proteins was quantitated by sandwich ELISA specific for the synapse protein as described in Example 1.5. Purified synapse proteins were spiked in a CSF pool and incubated overnight at 37 ° C (Legend: Rab3a in CSF; SNAP25 in CSF). As a control, the synapse protein in the same CSF pool was spiked and directly quantified (Legend: Rab3a; SNAP25). Stability was also examined in 1% BSA (results not shown).

3 , Demonstration of the Specificity of the Antibody of Transduction Labs (Lexington, KY, U.S.A., Cat # S63320) for α-Synuclein. A: Coomassie stained gel; Western blot developed with a monoclonal anti-His antibody to reveal the expression of all products; C: Western Blot developed with the monoclonal antibody from Transduction Labs (Lexington, KY, USA). Lane 1: expressing E. coli, α-synuclein; Lane 2: expressing E. coli, β-synuclein; Lane 3: expressing E. coli, γ-synuclein; Lane 4: control lane with neuron-specific enolase-expressing E. coli; Lane 5: molecular weight standards; Lane 6: E. coli strain without a plasmid.

4 , Detection of α-synuclein in 200 ml CSF. The pooled CSF was separated by molecular weight and by isoelectric point via rotophore as described in Example 2.2. M: molecular weight markers; R1: pI3; R2 pI 4; R3 pI 4.5; R4: pI 4.5; R5: pI 5; R6 pI 5; R7: pI 5.5 .; R8; pI 6; R9: pI6: R10: pI 6.5; R11: pI 6.5; R12: pI 7; R13: pI 7; R14: pI 7.5.

5 , Amino acid sequence of α-synuclein and overlapping peptides used to map the monoclonal antibodies 3B5 and 9B6 to the carboxy-terminus of α-synuclein. The portion of the carboxy-terminus used to synthesize the peptides is in bold. The peptides recognized by the monoclonal antibodies and by a commercial antibody (clone 42, hedgehog, transduction labs, Lexington, KY, USA) are shown.

6 , Optical density obtained after the reaction of various carboxy-terminal α-synuclein peptides with antibodies 3B5, 9B6 and clone 42. The optical density was measured in an immunoassay as described in Example 2.4. The numbers on the X axis (1-12) correspond to the numbers in 5 shown peptides.

7 , Rab3a and tau levels in CSF of 32 AD patients, 20 patients with vascular dementia (MID) and 11 controls measured by a sandwich ELISA as described in Examples 1.6 and 3.1.

8th , Correlations between the CSF levels of Rab3a and SNAP25, Rab3a and β-amyloid _(1-42), and Rab3a and Tau in AD patients described in Example 4.1. The levels of Rab3a, SNAP25, tau and β-amyloid _(1-42) were measured in a sandwich ELISA as described in Examples 1.6 and 3.

9 , Tau values at diagnosis before treatment: 1. AML (3); 2. AML-CNS + (1); 3. Down AML (2); 4. myelodysplasia (2); 5. Other [(medulloblastoma (2), rhabdomyosarcoma (2), intracranial germinoma (1)]; 6. B-NHL (8); 7. Hodgkin's disease (3); B. Down NB ALL (1); 9 NB ALL (21); 10. NB ALL-Brahman syndrome (1); 11. NB ALL-CNS ⁺ (1); 12. NB ALL-VHR (4); 13. Controls (6) (number of patients ).

10 , Concentrations of neurological CSF markers tau (a), neuromodulin (b), β-amyloid _(1-42) (c) and NSE (d), serum LDH (e) and CSF WBC) (f) on day 1. 1 AML; 2. AML-CNS +; 3. Down AML / MDS; 4. myelodysplasia; 5. Chronic myeloid leukemia; 6. B-NHL; 7. Hodgkin's disease; Down NB ALL; 9. NB ALL; 10. NB ALL-Brahman syndrome; 11. NB ALL-CNS +; 12. NB ALL-VHR; 13. LCH, rhabdomyosarcoma, germinoma, medulloblastoma, choriocarcinoma; 14. Controls, retinoblastoma (healthy), haemphagocytosis (healthy HLH). The detection methods for the markers are as described in Example 3.

11 , The levels of neurological CSF markers tau (a), neuromodulin (b) and neuron-specific enolase (c) as a function of lumbar puncture (LP) count during chemotherapy of B-cell NHL patients. The number on the X axis corresponds to the LP number as shown in Table 7a. The detection methods for the markers are described in Example 3.

12 , Levels of neurological CSF markers tau (a) and neuromodulin (b) for 13 non-B-ALL patients in different stages of chemotherapy treatment. The detection methods for the markers are described in Example 3.

13 , Levels of neurological CSF markers tau (a), β-amyloid _(1-42) (b), neuromodulin (c), and neuron-specific enolase (d) as a function of lumbar puncture (LP) count during non-B chemotherapy -cell-ALL patients. The number on the X axis corresponds to the number of LPs as indicated in Table 7b. The detection methods for the markers are as described in Example 3.

14 , Levels of tau (a) and neuromodulin (b) as a function of the lumbar puncture (LP) count during chemotherapy of starting material patients. The number of X-axis corresponds to the number of LP, as indicated in Table 7c. The detection methods for the markers are given in Example 3.

15 , Individual levels of tau (a) β-amyloid _(1-42) (b) and neuromodulin (or growth-associated protein 43) (c) in individuals classified as neurological controls (Guilain-Barré) multiple sclerosis, etc. ) (1 CONT), persons with memory impairment (2 MEM), a patient with pre-symptomatic familial Alzheimer's disease (PS1) mutation) (3 PATH), a patient with familial Alzheimer's disease (PS1 mutation (4 FAD), Alzheimer's disease) Patients (5 AD) and patients with vascular dementia (6VAD) as described in Example 7. The level of β-amyloid _(1-42) was measured as described in Example 3. Data are expressed in pg / ml.

16 , Correlation between individual levels of tau and neuromodulin in 60 patients with Alzheimer's disease (AD) and 32 controls at the appropriate age. The levels of tau and neuromodulin were measured as described in Example 3.

17 , Correlation between individual levels of tau / nm and β-amyloid _(1-42) in patients with Alzheimer's disease (AD), Parkinson's disease (PARK) and control patients (CONT). The levels of tau, neuromodulin and β-amyloid _(1-42) were measured as described in Example 3.

EXAMPLES

Example 1: Detection of Rab3a and SNAP25 in CSF

1.1 Cloning of Rab3a and SNAP 25

Specific primers were used to amplify the coding sequence of Rab3a and SNAP25 from the Quick-Screen ^™ human cDNA library (Clontech, Palo Alto, Calif., U.S.A., K1003-1) with the manufacturer's supplied amplification protocol , Briefly: 35 cycles at 94 ° C for 45 sec, 60 ° C annealing for 45 sec, and extension at 72 ° C for 2 min with Taq polymerase (Stratagene, Amsterdam, Netherlands Cat # 600131). The program was carried out with an extension of the polymerase reaction at 72 ° C for 7 min. completed. The reactions were carried out with a Perkin-Elmer DNA Thermal Cycler (Model 480) (Überlingen, Germany). The sequence of the primers for amplification of Rab3a was based on the human Rab3a sequence (Zahraoui et al., 1989): ATG GCA TCG GCC ACA GAC TCG CGC TAT GGG (T _m = 76 ° C) for the ATG primer and CGCG TCTAG AGG CTC TCA GCA GGC GCA GTC CTG GTG CGG (T _m = 77 ° C) for the reverse primer. The sequence of primers for the amplification of SNAP25 was based on the human SNAP25 sequence (Zhao et al., 1994): ATG GCC GAA GAC GCA GAC ATG CGC AAT GAG (T _m = 75 ° C) for the ATG primer and CGCG CTAG ACA CTT AAC CAC TTC CCA GCA TCT TTG TTG (T _m = 59 ° C) for the reverse primer.

The PCR products were reamplified to a sufficient amount PCR product, with T4 DNA polymerase blunt-ended, split with XbaI and clipped into NcoI, XbaI-digested pIGRHISA (Innogenetics, Gent, Belgium; 2075). This gave pIGRHISARab3a (Innogenetics, Gent, Belgium; Cat 3008) for Rab3a and pIGRH6SNAP25a (Innogenetics, Ghent, Belgium, cat # 2941) for SNAP25. The ligated product was transformed into DH1 (λ) (Bachman, 1987), and tetracycline resistant colonies were based on the presence of a Inserts examined. The inserts were sequenced, and the plasmids with the correct sequence (Zahraoui et al., 1989, Zhao et al., 1994) were still used. For Rab3a had multiple PCR artifacts. The correct sequence was assembled from two clones.

1.2 Expression and purification from Rab3a and SNAP25

Rab3a and SNAP were expressed in E. coli with a PL-based expression system pIGRHISARab3a and PIGRH6SNAP25a, respectively. The correct plasmid was identified in MC1061 pACI (Wertman et al., 1986) with a thermally sensitive cI repressor transformed. A Coomassie stainable band of 25 kDa was visible on a 12.5% acrylamide gel, indicating a reliable level of expression. The recombinant proteins were prepared as a fusion protein with 6 additional histidine residues, allowing rapid purification over NiIMAC columns. More than 10 mg of recombinant Rab3a and SNAP25 were purified to at least 95% homogeneity using 3 liters of heat-induced E. coli (Hochuli, 1988, Van Gelder et al., 1993).

1.3 Generation and Characterization of Rab3a and SNAP specific antibodies

Antibodies to recombinant Rab3a and SNAP25 were raised in rabbits. 50 μg of purified protein was injected intraperitoneally into 2 rabbits (100 μg / rabbit). Injections were made every 4 weeks and titers were determined by ELISA. The antibodies were characterized on brain extracts. The results for Rab3a immunoreactivity are as in 1 shown. Tissue samples from patients with Alzheimer's disease and control patients were prepared by rapid homogenization in 5 volumes of 1% SDS, 1% sodium vanadate, 10 mM Tris pH 7.4 and boiling in a water bath for 5 min. produced. The homogenates were SNAP25 for 5 min. The ligated product was transformed into DH1 (λ) (Bachman, 1987) and tetracycline resistant colonies were assayed for the presence of an insert. The inserts were sequenced and the plasmids with the correct sequence (Zahraoui et al., 1989; Zhao et al., 1994) were further used. For Rab3a, several PCR artifacts were present. The correct sequence was assembled from two clones.

1.2 Expression and purification from Rab3a and SNAP25

Rab3a and SNAP were in E. coli with a PL-based expression system pIGRHISARab3a and PIGRH6SNAP25a, respectively. The correct plasmid was in MC1061 pACI (Wertman et al., 1986) with a thermosensitive cI repressor transformed. A Coomassie stainable band was around 25 kDa visible on a 12.5% acrylamide gel, which is a reliable Expression level indicated. The recombinant proteins were named as Fusion protein with 6 additional Histidine residues prepared so that rapid cleaning over NiIMAC columns was possible. More than 10 mg of recombinant Rab3a and SNAP25 were added to at least 95% homogeneity by means of 3 liters heat-induced E. coli (Hochuli, 1988; Van Gelder et al., 1993).

1.3 Generation and Characterization of Rab3a and SNAP specific antibodies

Antibodies to recombinant Rab3a and SNAP25 were raised in rabbits. 50 μg of purified protein was injected intraperitoneally into 2 rabbits (100 μg / rabbit). Injections were made every 4 weeks and titers were determined by ELISA. The antibodies were characterized on brain extracts. The results for Rab3a immunoreactivity are as in 1 shown. Tissue samples from patients with Alzheimer's disease and control patients were prepared by rapid homogenization in 5 volumes of 1% SDS, 1% sodium vanadate, 10 mM Tris pH 7.4 and boiling in a water bath for 5 min. produced. The homogenates were centrifuged for 5 minutes (12000 x g, room temperature) to remove insoluble material. A small aliquot of the supernatants was used to measure protein concentration by the BCA method (Pierce, Rockford, Ill., USA). The supernatants were diluted with water to a protein concentration of 2 mg / ml and an equivalent volume of 2X sample buffer (250 mM Tris pH 6.8, 3% SDS, 10% glycerol, 0.006% bromophenol blue and 2%. (β-mercaptoethanol) was added, followed by Laemmli gel electrophoresis on 10 to 12% gels The proteins were radiolabeled on nitrocellulose (Schleicher and Schuell, Dassel, Germany, cat # 401196) with a semi-dry blotting. The nitrocellulose filter was blocked with 1% BSA in 10 mM Tris pH 7.5, 150 mM NaCl. Primary and secondary antibodies were added at appropriate concentrations (± 1 μg / ml) in 1% BSA.

1.4 Imunoaffinity purification the specific Rab3a- and SNAP25 antibody

Purified recombinant antigen was dialyzed overnight in 0.3 M NaHCO ₃ pH 8.6. The OD ₂₈₀ values were determined before and after dialysis to calculate the amount of protein. A mini-leak medium (KEM-EN-TEC-Biozyme, Vancouver, BC, Canada, Cat # 10127, Lot # 60232-5) was used to immunopurify the antibodies according to the manufacturer's instructions. Part of the antibodies were biotinylated (Amersham, Place Little Chalfont Buckinghamshire UK, cat # RPN 2202). Purification and labeling were monitored by silver staining and Western blotting (results not shown).

1.5 Assessment of Stability and the Present of Rab3a and SNAP25 in CSF

With the aid of specific monoclonal antibodies (Transduction Labs, Lexington, KY, USA; R35520 and 535020) as catcher antibodies and the immunopurified rabbit polyclonal antiserum as detection antibodies, the stability of the spiked synapse proteins Rab3a and SNAP25 in CSF was observed after incubation overnight at 37 ° C determined. At concentrations of 500 pg / ml, the recombinant immunoreactivity for SNAP25 and Rab3a changed overnight in the CSFs studied ( 2 ) Not.

a direct evidence that both proteins were present in CSF was by extraction of albumin and IgG from 50 ml CSF and column fractionation receive. The fractions were dried and dissolved in sample buffer a 12% acrylamide gel and blotted. The PVDF membranes were probed with monoclonal Rab3a and SNAP25 antibodies. Immunoreactive Bands of expected molecular weight and pI were determined.

1.6 Development of a Sandwich ELISA for the detection of Rab3a and SNAP25 and quantitative determination of CSF levels.

A sandwich ELISA based on a monoclonal Rab3a or SNAP25 antibody as a capture antibody and a biotinylated immunoaffinity purified polyclonal antibody as a detection antibody was developed. Maxisorp microtiter plates were coated with Affini-Pure Goat anti-Mouse IgG (Jackson Immuno Research Laboratories, Inc. West Grove, Pennsylvania, U.S.A., Cat # 115-035-144). 1% BSA (Clinical Grade, 98% Fatty Acid Free; ICN, Biomedical Research Products, Costa Mesa, Calif., USA, Cat # 105033, Lot # 6p384) in PBS was used as a blocking buffer. Mouse anti-Rab3a (Transduction Labs, Lexington, KY, USA; cat # R35520, IgG2a, clone 9, lot # 606-259-1550 lot 2) or anti-SNAP25 (Transduction Labs, Lexington, KY U.S.A., cat # S35020, hedgehog, clone 20), diluted 1/1000 in blocking buffer, was added. After incubation, recombinant antigen was added at various concentrations (concentration range 40000- 2.56 pg / ml). Simultaneously, the affinity-purified rabbit anti-Rab3a or anti-SNAP25 antiserum was added at a concentration chosen for optimal background signal ratio. The biotinylated rabbit antibodies were then detected on horseradish-labeled streptavidin (Immuno Research Laboratories, Inc. West Grove, Pennsylvania, USA, cat # 016-030-084) at 1/2000 dilution. Coupled peroxidase was detected via TMB, H ₂ O ₂ substrate solution. The reaction was stopped after 30 minutes with 2N H ₂ SO ₄ and the absorbance was measured at 450 nm. On the basis of this ELISA Rab3a could be detected in a concentration of only 10 pg / ml. The test for SNAP25 was based on the same principle.

Example 2: Presence and detection of α-synuclein in cerebrospinal fluid

2.1 Assessment of a commercial antibody on his specificity for α-synuclein

The specificity of a commercially available monoclonal antibody (Transduction Labs, Lexington, KY, USA, Cat # 563320, hedgehog) on the various synuclein isoforms was evaluated. Open reading frames of α-synuclein, β-synuclein and γ-synuclein (from the ATG to the stop codon) were isolated from a human brain cDNA library (HL5018, Clontech, Palo Alto, CA, USA) with primers based on the published sequence data ( α-synuclein: accession number L08850; β-synuclein: accession number S69965; γ-synuclein: accession number AF010126). There were reported to be some important amino acid changes in the original sequence of γ-synuclein: K12E and K68E, and the polymorphism of amino acid 109 in this clone is E109V. The insert was subcloned into a PL-based expression system (ICCG3307, Innogenetics, Ghent, Belgium) with the addition of 6 additional histidines at the amino terminus. His-end Synuclein fusion proteins were expressed in E. coli. The E. coli proteins were then separated by SDS-PAGE and immunoblotted with the commercial monoclonal antibody from Transduction Labs (Lexington, KY, USA). This monoclonal antibody was found to be specific for α-synuclein and mapped the carboxy-terminal half of the synuclein protein ( 3 ).

2.2 Assessment of the presence vopn α-synuclein in cerebrospinal fluid

CSF was unified by several patients. 200 ml of combined CSF were separated via rotophore according to the isoelectric point. Subsequently, the resulting fractions were separated by SDS-PAGE and immunoblotted with the monoclonal antibody from Transduction Labs (Lexington, KY, USA). Two immunoreactive bands were detected in the 19 kDa region and with a pI in the range of 4 to 6 ( 4 ). The lower band could be a C-terminal truncated form since the deletion of some negatively charged amino acids (6E in the last 20 amino acids) results in a shift of the pI to the more basic end. On the basis of this immunoreactivity, the concentration range of α-synuclein in the pg / ml range was recorded.

2.3 Generation and Characterization of α-synuclein-specific antibodies

α-synuclein was purified from 3 liters of induced E. coli cultures resulting in Purify more than 10 mg of α-synuclein (more than 90% pure, calculated with Coomassie and silver stained gels). The protein was in mice injected according to several immunization schemes (Table 3). To In 4 injections, the titer was evaluated against α-synuclein in a coating ELISA. six Had mice a titer over 100,000 (Titer defined as serum dilution at an OD value twice the background).

Three Days after the last injection, spleen cells from animal 312 (m3) recovered and used for cell fusion, mainly after the method as von Köhler and Milstein (1975). In a first round of surveys was hybridomas to the presence of specific antibodies in examined a direct coating test. Subsequently were They examined on a dot blot of E. coli lysates, the α-, (β- and γ-synuclein and deletion mutants of α-synuclein so as to select for hybridomas that produce antibodies, that recognize a different epitope on the synuclein protein.

2.4 Characterization of α-synuclein-specific antibody

Two hybridomas, 3B5 (IgG2a) and 9B6 (hedgehogs) were isolated, which, according to dot blots, were specific for α-synuclein. To determine their precise epitopes, the carboxy terminal portion (position 64 to 140) was determined to be 10 overlapping peptides ( 5 ). Such peptides were 14 amino acids long and the overlap was 7 amino acids. The biotinylated peptides were captured on streptavidin-coated plates at a concentration of 1 μg / ml. α-Synuclein-specific antibodies were incubated on these peptides and subsequently detected with anti-mouse enzyme-linked antibodies. The enzyme, horseradish peroxidase, was quantified using trimethylbenzidine as a substrate. 3B5 and 9B6 recognized the peptide containing the sequence LEDMPVDPNDNEAYE (position 113-126), suggesting a linear epitope for these monoclonal antibodies ( 6 ). In the same set of experiments, a commercial antibody (clone 42, IgG1, Transduction Labs, Lexington, KY, USA, cat # 563320) previously shown to recognize α-synuclein could also be targeted to a linear epitope be mapped (sequence AGSIAAATGFVKKD, position 85-98) ( 6 ).

2.5 Purification and Biotinylation the α-synuclein-specific antibody

To In a second and third round of subcloning, the production scale of the antibody For the purification of 10 to 20 mg of antibody increased to 1 to 2 liters. One Mini Leak Medium (KEM-EN-TEC Biozyme, Vancouver, BC, Canada, Cat. 10127, Batch no. 60232-5) was used for immunopurification of the antibodies according to the Manufacturer supplied information used.

The Biotinylation was carried out according to well-recognized methods (Bonhard et al., 1984) with D-biotinyl-etaaminocaproic acid N-hydroxysuccinimide ester (Boehringer-Mannheim, Brussels, Belgium, cat. No. 1008960).

2.6 Development of a Sandwich ELISA for the detection of α-synuclein and quantitative determination of levels in the cerebrospinal fluid

A sandwich ELISA based on an α-synuclein antibody as a capture antibody and one of the biotinylated monoclonal antibodies as a detection antibody is being developed. Maxisorp microtiter plates were coated with Affini-Pure Goat anti-Mouse IgG (Jackson Immuno Research Laboratories, Inc. West Grove, Pennsylvania, U.S.A., Cat # 115-035-144). 1% BSA (Clinical Grade, 98% Fatty Acid Free; ICN, Biomedical Research Products, Costa Mesa, Calif., USA, Cat # 105033, Batch # 6p384) + 1% mouse serum in PBS was used as a blocking buffer. Anti-α-synuclein (Transduction Labs, Lexington, KY, USA), diluted 1/1000 in blocking buffer, was added. After incubation, recombinant antigen was added at various concentrations (concentration range 1000-2 pg / ml). Concurrently, the biotinylated monoclonal anti-α-synuclein antibody was added in the presence of 1% mouse antibody in a concentration chosen for optimal background signal ratio. The biotinylated rabbit antibodies were then sequestered over horseradish peroxidase-labeled streptavidin (Immuno Research Laboratories, Inc. West Grove, Pennsylvania, U.S.A., Cat # 016-030-084) at a dilution of 1/2000 rejected. Coupled peroxidase was detected via TMB, H ₂ O ₂ substrate solution. The reaction was stopped after 30 minutes with 2N H ₂ SO ₄ and the absorbance was measured at 450 nm.

Example 3: Detection of others neurological marker in cerebrospinal fluid

3.1 Tau and Phospho-Tau

Total tau was measured with the Tau antigen test using AT120 as the capture antibody and biotinylated HT7-BT2 as detection antibody (INNOTEST hTau antigen, Innogenetics, Gent, Belgium). Monoclonal antibody AT120 reacts equally good with normal and hyperphosphorylated human tau protein (Vandermeeren et al., 1993), monoclonal antibody HT7 also responds equally well with normal and hyperphosphorylated human tau protein, whereas monoclonal antibody BT2 recognizes preferential normal tau (Goedert et al., 1994). affinity purified Tau protein, prepared as previously described (Mercken et al. 1992b), was used as standard.

Phospho-Tau was measured by a sandwich ELISA using HT7 as the capture antibody and biotinylated AT270 as detection antibody (INNOTEST Phospho-Tau (181), Innogenetics, Ghent, Belgium). AT270 detects specifically phosphao-tau (international application, published under WO 95/17429).

3.2 β-amyloid _(1-42)

β-amyloid _(1-42) concentrations were measured with Innotest β-amyloid _(1-42) (Innogenetics, Gent, Belgium). The assay is a sandwich ELISA using a first monoclonal antibody 21F2 (specific for the carboxy-terminus of amyloid) as the capture antibody and biotinylated 3D6 (specific for the amino terminus) as the detection antibody. The combination of 21F12 / 3D6 allows specific detection of β-amyloid _(1-42) peptide. Some cross-reactivity is observed for β-amyloid _(1-43) but not for shorter peptides (Citron et al., 1997, Johnson-Wood et al., 1997). Briefly, 21F12 antibody was suspended in 10mM Tris, 10mM NaCl and applied to Nunc Maxisorb's microtiter plates overnight at 4 ° C. After a wash, the plates were blocked for 2h at 25 ° C with PBS-0.1% casein. The assay was performed by simultaneous incubation (1 hr, 25 ° C) of 75 μl biotinylated 3D6 and 25 μl CSF or standard. After several washes, the amount of bound antibody was verified by addition of 100 μl of HRP-streptavidin (RDI, Flanders, New York, NY, USA). The incubation was continued for 30 min at 25 ° C. Then, 100 μl of 0.42 mM of 3,5,3 ', 5'-tetramethylbenzidine as a peroxidase substrate was added. The reaction was stopped after 30 min with 50 μl of 0.9 NH ₂ SO ₄ .

3.3 β-amyloid _(1-40)

The β-amyloid _(1-40) levels were measured with a C-terminal specific affinity purified polyclonal antibody from Quality Controlled Biochemicals (QCB, Hopkinton, MA, USA) as catcher antibody and 3D6 (Citron et al., 1997; Johnson-Wood et al., 1997) as detection antibodies. Nunc Maxisorp's microtiter plates were incubated for 2 hrs at 25 ° C with 5 μg / ml affinity-purified goat anti-rabbit IgG (H + L) (Jackson Immuno Research Laboratories, Inc. West Grove, Pennsylvania, USA, cat -005-144) in 10mM Tris-10mM NaCl buffer. Subsequently, the plates were blocked with PBS-0.1% casein overnight at 4 ° C. The polyclonal rabbit antibody (QCB, Hopkinton, MA, USA, cat # 44-348-20) was added at a concentration of 0.5 μg / ml for 1 h at 25 ° C. After several washes, 100 μl of CSF or standard was incubated for 2 hours at 25 ° C. The amount of amyloid bound was verified by adding 100 μl of biotinylated 3D6 antibody added at a concentration of 0.1 μg / ml in conjugate diluent, 1 h at 25 ° C. The plates were washed again five times. The amount of bound antibody was verified by addition of 100 μl of SV-AP (Gibco, Rockville, MD, U.S.A., JK-4410). The incubation was continued for 1 h at 25 ° C. After a final wash (five times), 100 μl of TMB dissolved in substrate buffer was added as the peroxidase substrate. The reaction was stopped after 30 min with 50 μl of 0.9 NH ₂ SO ₄ .

3.4 Neuromodulin

Neuromodulin was also measured by a sandwich ELISA using two epitope-specific monoclonal antibody (NM2, NM4; Oestreicher et al., 1994). NM2 was as catcher antibodies and biotinylated NM4 used as a detection antibody. recombinant Neuromodulin was used as standard.

3.5 Neuron-specific enolase

NSE measurements based on a sandwich ELISA with a monoclonal anti-NSE antibody, 2E7, as a catcher antibody and the peroxidase-labeled monoclonal anti-NSE antibody 10Cl as a detection antibody. Purified human brain NSE was used as standard (Vanmechelen et al., 1997).

The Protein concentrations were measured with the BCA protein reagent (Pierce, Rockford, Illinois, USA).

Example 4: Combination test using CSF-Rab3a, CSF-SNAP25, CSF-tau and CSF-β-amyloid _(1-42) as neurological markers for specifically detecting Alzheimer's disease and distinguishing Alzheimer's disease from controls in the same Age.

4.1 Patients and control individuals

The Alzheimer's disease group (AD) contained 32 patients, and indeed 15 men and 17 women, with a mean age ± SD of 75.0 ± 6.6 years. The vascular Dementia group (VAD) included 20 patients, 10 men and 10 women with one middle age ± SD from 81.0 ± 7.0 Years. The control group contained 18 individuals, 7 men and 11 women, with a mean age ± SD of 67.5 ± 5.5 years. The diagnosis of possible AD was made by exclusion according to the NINCDS-ADRDA criteria (McKhann et al., 1984). VAD has been reported in patients with transient ischemic Attacks and / or stroke episodes in relation to development of dementia and / or CT findings of large infarcts and / or multiple Lacunae and / or a history or clinical findings of severe vascular Diseases such as arterial hypertension or diabetes mellitus Complications diagnosed. The control group consisted of individuals without stories, symptoms or signs of psychiatric or neurological disease, malignant disease or systemic disorders (for example rheumatoid arthritis, infectious disease). In individuals above one At age 60, the cognitive state was assessed with the mini-mental state examination (Folstein et al., 1975). Individuals below 28 were not added. The study was conducted by the Ethics Committee of the University of Gothenburg (Gothenburg, Sweden). The patients (or their closest relatives) and the Individuals of the control group gave their informed consent to participate in the investigation.

4.2 Isolation of brain-specific cerebrospinal fluid

The method has been described in detail by Davidsson et al., 1996. In summary, 5 to 10 ml of CSF were applied to a Blue Sepharose column (Pharmacia, Uppsala, Sweden) for the selective removal of albumin. The albumin-free fraction was then applied to a staphylococcal protein G column covalently bound to Sepharose 4B (Pharmacia, Uppsala, Sweden) so that IgG was absorbed. The unabsorbed proteins were analyzed by mR-HPLC by SMART system (Pharmacia LKB-Technology, Uppsala, Sweden) with a mRPC C ₂ / C ₁₈ column (Abm i i D. 2.1 x 100 mm, gel volume 0.35 ml, particle size 3 mm). The proteins were eluted with two linear gradients of trifluoroacetic acid. A total of 40 fractions were dried in a Savant Speed Vac concentrator. The fractions were dissolved in an SDS-PAGE sample buffer, sonicated, and boiled for 15 min prior to separation on 12% polyacrylamide gels.

4.3 Assessment of a combination test for the specific evidence of Alzheimer's disease

Using a sandwich ELISA, levels of CSF-Rab3a, CSF-SNAP25, CSF-tau, and CSF-β-amyloid _{(1-42) were} measured in the CSF samples from 32 AD patients, 20 VAD patients, and 11 controls measured. The averaged values for all markers are summarized in Table 4. The individual values for Tau and Rab3a are in the 7 specified.

Probability ratios were determined based on an exclusion threshold determined by the highest sensitivity for AD patients and the highest specificity for the control patient (Table 5). Since the Rab3a and SNAP25 values are correlated, one can only use either Rab3a or SNAP25 in combination with Tau and β-Amyloid _(1-42) ( 8th ). The probability ratios for the combinations of Rab3a and SNAP25 were thus not determined. The increased probability ratios for the combination of the three neurological markers tau-β-amyloid _(1-42) -Rab3a and tau-β-amyloid _(1-42) -SNAP25 compared to the probability ratios for only one of the two respective markers show that these combinations of the three markers allow a more specific and sensitive detection of Alzheimer's disease.

A complete factorial multiple analysis of variance (ANOVA), with all markers as dependent variable, gender as factor, age and minimental score score (MMSE, Folstein et al., 1975) as covariate, within the different groups showed that none this parameter covariates. In addition, significant correlations between tau, β-amyloid _(1-42) and Rab3a / SNAP25 could be detected in each group individually or in all groups together.

Additional antibodies are isolated for Rab3a and SNAP25. Levels for CSF-Rab3a, CSF-SNAP25, CSF-tau, and CSF-β-amyloid _(1-42) are examined in well-defined clinical diganose groups, where acceptable sample size allows statistically significant comparisons.

Example 5: combination test, using CSF-tau, CSF-neuromodulin and CSF-neuron specific Enolase as a neurological marker for the diagnosis of chemotherapy-induced neuronal damage Cattle suffering from leukemia be treated.

5.1 patients and control individuals

Between August 1996 and September 1998, 448 CSF samples were taken from 83 Children admitted to the Pediatric Hemato-oncology Department of the Catholic University of Leuwen, Belgium, treated for cancer were. All Patients went through a careful clinical evaluation at the time of diagnosis. It was the informed Consent of the parents.

The Samples were only taken in the course of planned lumbar punctures (LPs) taken for staging or for the treatment of malignancy. Various group of patients with hematological malignancies were for the present investigation (Table 6). A first group included 9 B-cell non-Hodgkin's lymphoma patients (B-NHL), which according to the NHL protocol United Kingdom Children Cancers Group (UKCCSG 9602) were (Table 7a). In this group, four patients had B-cell lymphomas, 3 patients had Burkitt's lymphoma and 2 patients had one anaplastic large cell lymphoma (ALCL). Eight of these patients were examined longitudinally. The second and largest group consisted of 42 patients with acute lymphoblastic non-B-cell leukemia / non-Hodgkin's lymphoma (NB ALL / NHL), treated according to the protocol 58881 (Table 7b) of the 'European Organization for Research and Treatment of Cancer (EORTC). In this second group had 18 children CD10 (+) outbreaks or general NB-ALL, two Patients had Down syndrome (DS), 1 patient had Brachmann-Lange syndrome, 3 Patients had general B-cell outbreaks, 1 patient had general T-cell blasts, 2 patients had pro-B-cell outbreaks, 9 patients had pre-B-cell outbreaks, and 8 patients had T-cell outbreaks. 38 children had leukemia, 5 patients had stage II non-Hodgkin's lymphoma (1 patient), Stage III (3 patients) or Stage IV (1 patient), of which a patient has evident CNS involvement had (CNS +), defined according to the examination protocol with malignant cells in CSF, eye fundoscopy and contrast trapping. Five patients proved to be very high risk patients (VHR) according to the criteria which are defined in the treatment protocol (2 patients with T cell outbreak and 3 patients with corticoid resistance). 27 patients from this group could be longitudinal be followed. A third group of patients consisted of 6 children with acute myeloid leukemia (AML), of which 1 patient had CNS involvement and 2 patients Down syndrome exhibited. There were 3 patients with MO and 1 each with M1, M2 or M7 phenotype. All of these patients were in accordance with EORTC 58921 protocol (Table 7c) treated and longitudinal tracked. The other patients consisted of a group of children (n = 18), in which a lumbar puncture was used for clinical reasons Diagnosis of infection and staging during or performed after her treatment has been. This group includes 5 children with medulloblastoma (3 staging, 1 during the Treatment, 1 follow-up), 3 children with Hodgkin's disease (staging, 3 children with Rhabdomyosarcoma (2 staging, 1 during treatment), 2 children with myelodysplastic syndrome (MDS) (staging), 2 children with Histiocytis the Langerhans'schen Cells (LCH / HLH, staging), 1 child with juvenile chronic myeloid leukemia (CML) (during treatment), choriocarcinoma (follow-up) or germinoma (staging). A big Group of healthy newborns was unavailable because of the removal of the cerebrospinal fluid out of these patients is unethical. As controls were children who are suspected of having meningitis but with negative findings (n = 4), a localized retinoblastoma (n = 1) and familial hemophagocytic Lymphohistiocytosis (n = 1). The informed consent the parents were received.

5.2. study design

One prospective and longitudinal single center examination design was used. No patient received treatment before entering the study. In the present explorative study, CSF samples were used of 58 children available before any treatment (= diagnostic lumbar puncture or LP1) (see Table 7 for more details). Residual samples were at most times for most Patient not present. Missing values are not based on the Disease status of children, but on the result of artifacts while sampling or storage of samples.

The Lumbar punctures were done for Routine analysis either at the baseline for diagnostic preparation or right before the IT administration of chemo. 5 ml of CSF was collected in different polypropylene tubes. A sample was immediately at 1500 rpm for Centrifuged for 2 minutes to eliminate cells and other insoluble material. The supernatant was for the subsequent one Stored at -70 ° C. The number of freeze-thaw cycles was minimized. The Routine CSF measurement included cytology, protein concentration, glucose, etc.

There the normality for all neurological marker data, regardless of the diagnostic groups, sorted out was, were for the analysis is non-parametric Statistics used. The Kruskall-Wallis test was used to study Uses group differences that affect the effect variables (Tau, neuromodulin, protein, etc.). Matching couples in the Wilcoxon rank sum test, were up for review Differences between the first diagnostic LP and subsequent LPs used. The Pearson correlation was used to study the possible Covariate used. The analyzes were done with the Prism software v2.01 (Graphpad Software Inc. San Diego, CA, USA) Systat version 7 (SPSS, Chicago, IL, USA).

5.3 Assessment of a combination test for the diagnosis of chemo-induced neuronal damage.

Mirror of the neurological Markers before treatment

normal Mirror the upper limit for Tau were first tested on CSF samples from children with infectious diseases (but with negative virus and bacterial cultures) (n = 4), one Patient with a very localized retinoblastoma and a patient, the on familial HLH was examined. The tau averages in the control children were 106.2 pg / ml (95% C2 = 34.3-178.0). An arbitrary limit to normal values became was considered to be 312 pg / ml (mean + 3 SD) in the range of Values that are observed in adults. 80 ° s of normal Adult controls have tau values below 352 pg / ml, while 425 pg / ml (p25-p75; 274-713, n = 150) mean tau values in patients with Alzheimer's disease were (Hulstaert et al., 1999). The samples were initially independent of the number of patients is examined, after which all samples taken from one Patients were re-examined on an immunoplate. The correlation coefficient between the results from the first and second approach for a set of 104 samples was 0.901 (95% CI: 0.859-0.933).

The tau levels at diagnosis were analyzed for each subgroup of patients ( 9 ). The tau levels at diagnosis ranged from 66 to 1500 pg / ml. The data for tau, neuromodulin, β-amyloid _(1-42) , β-amyloid _(1-42) , serum LDH, and the white blood cell count in CSF ( 10 ) show no obvious difference in the patient groups with and without Down syndrome or between the diagnosis groups. In addition, no correlation was found between the tau level and the WBC or LDH levels. There was no significant correlation between the tau concentration and the age of the children. The two patients with MDS, patients with overt CNS invasion (CNS +), but no children with AML had significantly increased tau levels at diagnosis. Also, three hospitalized patients with increased intracranial pressure due to medulloblastoma in the posterior fossa from which CSF staging was obtained had very high CSF-tau concentrations (823, 1387, 1500). 7/21 children with non-B ALL / NHL, 1/4 non-B ALL / NHL patients with very high-risk conditions, 2/4 patients with AML and 2/8 patients with B-cell NHL had a tau level above 312 pg / ml, although CNS invasion could not be detected by classical diagnostic methods.

For the 27 patients with non-B ALL / NHL or nine patients with B-cell NHL (data not shown), tau levels at diagnosis did not correlate with tumor burden, as determined by the number of white blood cells (p = 0.935, n = 40) or serum LDH (p = 0.855, n = 39). In LP1, there was a very significant correlation between tau and neuromodulin [r = 0.793; 95% CI: 0.658-0.928, n = 50), indicating that the secretion of both proteins is linked at some point. There was no correlation between tau and β-amyloid _(1-42) (p = 0.1032, n = 52).

Mirror of the neurological Marker in the treatment of B-NHL patients

The most striking Differences in chemotherapy-induced increases of tau in ng / ml, 3.4 ng / ml); LP2 (11.7 ng / ml, 9.6 ng / ml)] a noticeable one Increase for NSE observed.

Analysis of all data for the non-B-ALL patients revealed that the highest tau concentrations were observed during the induction period. During this induction period, 41% of the analyzed samples (28/68) were higher than 500 pg / ml. This percentage then dropped to 18.9% (14/74) in the interval period, to 16.7% (3/18) during the reinduction and finally to 9.7% (7/72) in the maintenance period ( 13a ). The tau values in the three patients who already had elevated tau (> 500 pg / ml) before starting the treatment were normalized after chemotherapy. The data for β-amyloid _(1-42) , neuromodulin and NSE levels in the non-B-ALL patients are in the 13b . 13c respectively. 13d shown. There was also a striking correlation between the levels of tau and neuromodulin (r = 0.658, 95% CI = 0.580-0.725, n = 251) or NSE (r = 0.589, 95% CI = 0.363-0, 749, n = 47).

Five non-B-ALL children with very high risk criteria were examined longitudinally in the present study. The first phase the treatment was similar EORTC 58881. Similar chemotherapy-induced changes The tau levels were in 4 out of 5 patients along with a very significant correlation between tau and neuromodulin (n = 55; r = 0.880, 95% CI: 0.802-0.929).

One Patient with Down syndrome non-B-ALL had a tau concentration the diagnosis of 70 pg / ml. The mean increase of dew in all Non-B-ALL patients were at 250% (95% CI = 130% -370%, n = 13) Day 8 and 200% (95% CI = 150% -260%, n = 8) on day 21 in the longitudinal study, the percentage increase of tau in the patient with Down syndrome 820% and 1200% on days 8 and 21, respectively.

One Certain patient was treated with prednisolone for 8 days without IT MTX a day 1 due to a high leukemic Debt (610000 WBC / mm3). After 8 days treatment remained the dew levels low, namely 155 pg / ml. Subsequently This patient entered the EORTC protocol like all other non-B-ALL patients, including IT injections from MTX during the next Weeks. The tau levels rose rapidly to 744, 948, 1120, 861 and 1023 pg / ml on day 12, 15, 18, 22 and 44, respectively. A CSF sample was available from a child who had been treated in another center, and the manifest neurotoxicity after treatment with MTX suffered and had diplegia. The tau and neuromodulin levels in exceeded this child the highest used standard (1500 pg / ml for tau, 8000 pg / ml for neuromodulin), which reflects the gross neuronal degeneration.

Mirror of the neurological Markers during the treatment for AML

The 14a shows the evolution of tau in individual patients with AML. Patients 11, 12, and 23 had no significant increase in tau during treatment (Table 7c). Patient 12, from whom the LPs were taken on days 1 and 8, had an aggressive disease and died early after bone marrow transplantation. Longitudinal data were available from one of the two patients with Down syndrome, and this patient had tau levels slightly above 300 pg / ml, in contrast to the evolution of tau levels in the CSF of patients 11 and 23. Patient 69 with proven CNS invasion at diagnosis had a tremendous increase in tau and neuromodulin ( 14b in CSF. This child is still being treated, and is currently in a state of complete remission.

conclusion

We found in our longitudinal study significant increases in the levels of tau, neuromodulin and NSE in the CSF, most likely a chemo-related neural damage Mirroring, and primarily at the time of IT MTX in combination with IV corticosteroids and chemotherapy (ALL induction and reinduction therapy, B-cell NHL therapy) were induced, but not during High-dose IV and IT-MTX in interval therapy.

Example 6: Rombination test to diagnose the brain damage of a perinatal asphyxia arises.

Perinatal asphyxia can be associated with neuronal damage. In addition to the electroencephalographic and neuroradiological data, the CSF neurological markers may contribute to clinical data complement the evaluation of hypoxic-ischemic events (Garcia-Alix, 1994). It has become increasingly apparent that modified brain metabolic activity is reflected by changes in the components in the CSF. The perinatal levels of CSF markers and the distribution of changes due to asphyxia are assessed.

Example 7: Combination test using CSF neuromodulin, CSF-tau and CSF-β-amyloid _(1-42) as neurological markers for specific detection of Alzheimer's disease and for distinguishing Alzheimer's disease from control individuals

7.1 Patients and control individuals

CSF was obtained from 109 individuals. The individuals were in four Divided into groups: (i) a group called neurological controls for dementia is defined (n = 70). This group includes patients with multiple Sclerosis, Guillain-Barre syndrome, polyneuropathy and epilepsy. The other groups consisted of (ii) elderly patients with memory impairment (n = 7), (iii) patients with Alzheimer's disease (AD) (n = 27) and (iv) Patients with vascular Dementia (VAD) (n = 5). All Patients were diagnosed according to the criteria those of the International Classification of Diseases, Version 9, (Handbook of International Classification of Diseases, Injuries and causes of death: based on the recommendations of the Ninth Review Conference, 1975; and adapted from the 29th World Health Assembly, 1977) were determined. In the AD group, 2 patients came from one Family in which a presenilin mutation has an early onset of the disease. One of these patients was presymptomatic.

CSF was routinely taken as part of the neurological examination. All tests were done on the rest of the CSF. β-Amyloid _(1-42) was measured only in CSF samples that were stored correctly in polypropylene tubes and frozen only once.

7.2 Evaluation of a combination test for the specific detection of Alzheimer's disease and the distinction of individuals with Alzheimer's disease versus control individuals

The levels of tau, β-amyloid _(1-42) and neuromodulin (GAP-43) in the CSF of these patients were determined. The mean CSF levels for each group are shown in Table 8. Individual levels of β-amyloid _(1-42) , tau and neuromodulin are as in the 15a . 15b and 15c shown.

CSF-tau and CSF-β-amyloid _(1-42) were significantly altered in AD patients. The CSF neuromodulin was significantly increased in AD. In the elderly patients with memory disorders, however, no significant effect was observed in CSF neuromodulin, whereas the CSF-tau level was significantly increased compared to control individuals. Three of the 7 patients with memory impairment had CSF tau levels above the maximum value defined by the control group (280 pg / ml). Due to incorrect storage, CSF-β-amyloid _(1-42) levels could only be determined in 3 patients with memory impairment. Levels below 300 pg / ml were found in two of the three patients. One of these patients also had an elevated CSF-tau level. The other patient had a CSF tau level of 278 pg / ml, ie just below the maximum defined by the control group. In the patient with presymptomatic familial AD, an increase in CSF tau levels of 327 pg / ml was observed, with a β-amyloid _(1-42) level below 300 pg / ml. This analysis on a few samples from individuals with memory impairment indicate that the combined use of tau, β-amyloid _(1-42) and neuromodulin is a better indicator of the presence of Alzheimer's disease. For statistical significance, a larger number of samples per group is examined (at least 50 samples per diagnostic group).

Example 8: Combination test using CSF neuromodulin, CSF-β-amyloid _(1-42) and CSF-tau as neurological markers for the specific detection of Alzheimer's disease, to distinguish individuals with Alzheimer's disease from control individuals, for the specific Detection of Parkinson's disease to distinguish individuals with Parkinson's disease from control individuals and to distinguish between Alzheimer's disease and Parkinson's disease.

8.1 Patients and control individuals

CSF was obtained from 60 patients with Alzheimer's disease, 23 patients with Parkinson's disease and 32 controls at the appropriate age. The levels of tau, β-amyloid _(1-42) and neuromodulin (GAP-43) in the CSF of these patients were determined. The mean CSF levels for each group are shown in Table 9.

8.2 Evaluation of a combination test for the specific detection of Alzheimer's disease and for differentiation between individuals with Alzheimer's disease and control individuals

Tau was significantly elevated in the AD patients compared to the controls, whereas the β-amyloid _(1-42) levels were lower. NM levels were not significantly different between the two groups. However, the NM and tau levels correlated significantly ( 16 ). By watching the 16 shows that the values for the AD patients could be separated from the control patients. A reverse discriminant analysis (Morrison, 1976) was used to determine which variables, tau, β-amyloid _(1-42) , neuromodulin, or tau / neuromodulin, could best distinguish between patients with Alzheimer's disease and control patients. The variables tau / neuromodulin and β-amyloid _(1-42) were selected and correctly classified 55 out of 58 patients with Alzheimer's disease (sensitivity 94.8, CI 85.6% -98.9%) and 30 of 31 control patients (Specificity 96.8%, CI 83.3% -99.9%) ( 17 ). Further analysis of a larger number of samples allows a statistically significant improvement in the diagnosis of Alzheimer's disease by using these three markers.

8.3 Evaluation of a combination test for the specific detection of Parkinson's disease and for differentiation of individuals with Parkinson's disease versus control individuals.

A backward discriminant analysis (Morrison, 1976) was used to determine which variables could best distinguish between control and Parkinson's disease patients. The variables tau / neuromodulin and β-amyloid _(1-42) were selected. 14 of the 23 patients with Parkinson's disease (sensitivity 60.9%, CI 38.6-80.3) and 27 of the 31 control patients (specificity 87.1%, CI 70.2% -96.4%) were correctly classified ( 17 ). Further analysis of a larger number of samples allows a statistically significant improvement in the diagnosis of Parkinson's disease by using these three markers.

8.4 Assessment of a combination test to distinguish between Alzheimer's disease versus Parkinson's disease.

β-amyloid _(1-42) and neuromodulin were used to distinguish between patients with Alzheimer's disease and patients with Parkinson's disease with a sensitivity of 94.8% (CI 85.6% -98.9%) for those with Alzheimer's disease Disease and a specificity of 78.3% (CI 56.3% -92.5%) for patients with Parkinson's disease. Further analysis of a larger number of samples allows a statistically significant improvement in the distinction between Alzheimer's disease and Parkinson's disease by using these three markers.

Example 9: Use of CSF-α-synuclein as a neurological marker to distinguish between Lewy body dementia and Alzheimer's disease

9.1 Patients and control individuals

CSF is made up of 30 to 40 patients with Alzheimer's disease, 10 to 20 patients with Lewy body dementia and get 10 to 20 controls at the appropriate age.

9.2 Differential diagnosis test from Alzheimer's disease and Lewy body dementia

The concentration of α-synuclein in the CSF of the different patients and control groups is quantified. A significantly different mean for the α-synuclein level in the Alzheimer's disease group versus the mean for the α-synuclein level in the Lewy body dementia group allows the distinction between these two types of dementia. The distinction between Alzheimer's disease and Lewy body dementia is further enhanced by combining the quantification of α-synuclein with the quantification of at least 2 other neurological markers (such as tau and β-amyloid _(1-42) ).

Example 10: Combination test using CSF-β-amyloid _(1-42) , CSF-tau and CSF-phospho-tau as neurological markers for the specific detection of dementia of the frontal lobe / temporalis and for distinguishing the dementia of the frontal lobe / temporalis over another dementia

10.1 patients and control individuals

CSF was obtained from 30 to 40 patients with dementia of the lobe frontalis / temporalis and 10 to 20 controls in the corresponding Age.

10.2 combination test for the Differential diagnosis of dementia of the frontal lobe / temporal lobe compared to control individuals

The level of β-amyloid _(1-42) , tau and phospho-tau of various patients with frontal lobe / frontal lobe dementia and in control patients was quantified. The mean values for the levels of tau and phospho-tau in the patients with dementia of the frontal lobe are significantly increased compared to the mean values for the levels of tau and phospho-tau in the control patients. The patients with dementia of the frontal lobe / temporalis show a lower level of β-amyloid _(1-42) than the control patients.

Example 11: Combination Assay Using CSF-β Amyloid _(1-42) , CSF-Tau, and Plasma β-Amyloid _(1-42) as Neurological Markers for Specific Detection of Alzheimer's Disease Vascular Problems and Vascular Discrimination Illness from others. Forms of Alzheimer's disease

11.1 Patients and control individuals.

CSF was obtained from 30 to 40 patients with vascular disease, 30 to 40 patients with other forms of Alzheimer's disease and 10 to 20 controls at the appropriate age.

11.2 Rombination test for the differential diagnosis of vascular disease over others Forms of Alzheimer's disease

The levels of tau and β-amyloid _(1-42) in the CSF and the levels of β-amyloid _(1-42) in the plasma of the various patients with vascular disease, with other forms of Alzheimer's disease and in control patients were quantified , A quantitatively different level of plasma β-amyloid _(1-42) , CSF-tau, and β-amyloid _(1-42) allows differentiation between patients with vascular disease and patients with other forms of Alzheimer's disease.

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Claims (5)

A method for the specific detection, quantification and / or differential diagnosis of neurodegeneration in a patient comprising the step of determining the level of at least three neurological markers in one or more body fluid sample (s) of the patient or determining the level of the same marker in two various body fluid samples in combination with the detection of at least one other marker in one or more body fluid samples with antibodies specifically recognizing these neurological markers, the type and level of neurodegeneration being compared in a quantitative change in the level of all these neurological markers with a control, the method being further characterized by : - specifically detecting or quantifying Alzheimer's disease and / or Lewy body disease and / or differential diagnosis of Alzheimer's disease in contrast to Lewy body disease, the levels of tau, β-amyloid _(1-42) and α-synuclein are determined in a patient's cerebrospinal fluid sample; The specific detection or quantification of Alzheimer's disease and / or the differential diagnosis of Alzheimer's disease versus other dementia, the level of tau, β-amyloid _(1-42) and Rab3a in a patient's cerebrospinal fluid sample; or the level of the mirror on tau, β-amyloid _(1-42) and SNAP25 in a patient cerebrospinal fluid sample is true; For the specific detection or quantification of Alzheimer's disease and / or Parkinson's disease and / or for the differential diagnosis of Alzheimer's disease against Parkinson's disease, the levels of tau, β-amyloid _(1-42) and neuromodulin in a patient's cerebrospinal fluid sample is determined; For the specific detection or quantification of neurodegeneration induced by chemotherapy and / or exposure to chemical compounds and / or irradiation, the levels of tau, neuron-specific enolase and neuromodulin are determined in a patient's cerebrospinal fluid sample; For the specific detection or quantification of frontalis / temporal lobe dementia and / or for the differential diagnosis of dementia of the frontal lobe / temporal lobe against other dementia, the levels of tau, phospho-tau and β-amyloid _(1-42) in a sample of cerebrospinal fluid Patient is determined; - for the specific detection or quantification of vascular problems in Alzheimer's disease, for the differential diagnosis of different forms of Alzheimer's disease and / or for the differential diagnosis of Alzheimer's disease versus other dementia • the levels of tau and β-amyloid _(1-42) quantitatively in a cerebrospinal fluid sample and the level of β-amyloid _{(1-42) is determined} quantitatively in a patient's plasma sample, or • the level of phospho-tau and β-amyloid _{(1-42) is} quantified in a cerebrospinal fluid sample and the level of β- Amyloid _{(1-42) is} quantitated in a patient's plasma sample, or • the level of tau and phospho-tau is quantified in a cerebrospinal fluid sample and the level of β-amyloid _{(1-42) is} quantitatively determined in a patient's plasma sample, or the level of tau, phospho-tau and β-amyloid _{(1-42) was} quantified in a cerebrospinal fluid sample of the patient w ill. Method according to claim 1, further characterized that the patient whose chemotherapy, exposure to chemical Compounds and / or radiation induced neurodegeneration demonstrated or quantified, leukemia or brain tumor suffers. A diagnostic kit for the specific detection, quantification and / or differential diagnosis of neurodegeneration in a patient comprising at least 3 antibodies, each of which recognizes a different neurological marker and recognizes one of the following combinations of neurological markers: - tau, β-amyloid _{(1-) 42)} , α-synuclein; Tau, β-amyloid _(1-42) , Rab3a; Tau, β-amyloid _(1-42) , SNAP25; Tau, β-amyloid _(1-42) , neuromodulin; - Tau, neuron-specific enolase, neuromodulin or - tau, phospho-tau, β-amyloid _(1-42) ; or at least 2 antibodies, each of which recognizes a different neurological marker of which an antibody recognizes a neurological marker in 2 different body fluids and which recognizes one of the following combinations of neurological markers: tau, β-amyloid _(1-42) or Phospho-Tau, β-amyloid _(1-42) A diagnostic kit according to claim 3 for the specific detection, quantification and / or differential diagnosis of neurodegeneration in a patient, comprising: a carrier comprising, together or separately, at least 3 antibodies (primary antibody or capture antibody) each recognizing a different neurological marker; or comprises at least 2 antibodies (primary antibody or capture antibody), each of which recognizes a different neurological marker of which an antibody recognizes a neurological marker in 2 different body fluids; - secondary antibodies (detection antibodies), each of which recognizes one of the complexes of neurological marker-primary antibody; Optionally a marker for either the specific labeling of or coupling with the secondary antibodies; If appropriate, suitable buffer solutions for carrying out the immunological reaction between the primary antibodies and the body fluid sample, between the secondary antibodies and the neurological marker primary antibody complexes and / or between the bound secondary antibodies and the marker; Optionally protein purified for standardization or synthetic peptides specifically recognized by the antibodies of the kit used to detect the neurological marker the. Use of a method or a diagnostic Kits according to any one of claims 1 to 4 for therapeutic monitoring and / or determining the effectiveness of a particular treatment.